Financial support from DFG (projects SM 82/13-1) is gratefully acknowledged.

1. Preparation of Amidated Pectin Stock Solution
<ol>
	<li>Mix 20 g amidated pectin with 980 g water (2.0 wt%). The degree of amidation is 25 wt%.</li>
	<li>Homogenize the solution with a high speed stirrer (10,000 rpm) for 2 min to obtain a homogenous viscous solution.</li>
	<li>Measure pH using pH strips or pH meter. If the pH is lower than 6.5, titrate with 0.5 M NaOH to neutralize the solution (to pH 7.0).</li>
	<li>Add calcium carbonate in a ratio of 0.1825 g per gram of dry amidated pectin (q=1). &#34;q&#34; denotes the degree of crosslinking.</li>
	<li>For 1 kg 2.0 wt% amidated pectin solution, add 3.65 g calcium carbonate (q=1)7 per 20.0 g dry pectin.</li>
	<li>For greater crosslinking, add 0.3650 g calcium carbonate per gram of dry amidated pectin (q=2).</li>
</ol>
2. Production of Hydrogels
<ol>
	<li>Homogenize the amidated pectin/calcium carbonate mixture using the high speed homogenizer (10,000 rpm) to obtain a white homogenous dispersion.</li>
	<li>Transfer the suspension into open polypropylene molds or glass Petri dishes.</li>
	<li>Place the molds in the high pressure autoclave. Seal the autoclave.</li>
	<li>Pressurize the autoclave with gaseous CO2 up to 5 MPa at RT. Refer to Gurikov et al.7 for further information. Maintain the pressure for 24 hr.</li>
	<li>Slowly depressurize the autoclave at 0.2 MPa/min.</li>
	<li>Open the autoclave and remove the molds. Remove the hydrogels from the molds by turning them over. If necessary, use a spatula.</li>
</ol>
3. Solvent Exchange Procedure
<ol>
	<li>Prepare 10 g of 10:90 (w/w) ethanol/water mixture per gram of hydrogel.</li>
	<li>Immerse the hydrogels in the 10:90 (w/w) ethanol/water mixture for 12 hr.</li>
	<li>Continue this process with increasing ethanol concentrations, i.e., from 10:90 (w/w) ethanol/water mixture to 30:70 (w/w) ethanol/water mixture. After 12 hr, transfer to 50:50 (w/w) ethanol/water mixture, then to 70:30 (12 hr), then 90:10 (12 hr) and then to 100% ethanol solution (12 hr).</li>
	<li>Soak the gel further in pure ethanol so that the final concentration inside the gel is more than 98% (w/w). Measure the concentration using the density meter. The alcogel is now ready for supercritical CO2 drying.</li>
</ol>
4. Production of Aerogels by Supercritical CO2 Drying
<ol>
	<li>Place the samples in the same high pressure autoclave used for hydrogel preparation (see step 2.3).</li>
	<li>Fill the autoclave with additional ethanol (2-10% of the autoclave volume) to prevent premature solvent evaporation from the gels. Complete immersion of gel in the solvent is not required.</li>
	<li>Seal the autoclave. Turn on the autoclave heating. Set the autoclave working temperature to 323 K. Pressurize the autoclave with carbon dioxide to 12 MPa using a compressor or pump.</li>
	<li>Periodically replace the CO2 inside the autoclave10,11 with fresh CO2 keeping the pressure constant. 6-7 residence volumes are required over a period of 6 hr. Refer to Gurikov et al.7 for further information.</li>
	<li>Slowly depressurize the autoclave at 0.2 MPa/min.</li>
	<li>Open the autoclave and collect the aerogel. Store the aerogel in an exicator or a sealed container.</li>
</ol>

<ol>
	<li>Kistler, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coherent+expanded-aerogels.">Coherent expanded-aerogels.</a> J. Phys. Chem. 36 (1), 52-64 (1932).</li><li>Aegerter, M. A., Leventis, N., Koebel, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Aerogels Handbook. , (2011).</li><li>Raman, S. P., Gurikov, P., Smirnova, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+alginate+based+aerogels+by+carbon+dioxide+induced+gelation:+Novel+technique+for+multiple+applications.">Hybrid alginate based aerogels by carbon dioxide induced gelation: Novel technique for multiple applications.</a> J. Supercrit. Fluids. 106, 23-33 (2015).</li><li>Draget, K. I., Skj&#229;k-Br&#230;k, G., Smidsr&#248;d, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate+based+new+materials.">Alginate based new materials.</a> Int. J. Biol. Macromol. 21 (1), 47-55 (1997).</li><li>Garc&#237;a-Gonz&#225;lez, C. A., Alnaief, M., Smirnova, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysaccharide-based+aerogels-Promising+biodegradable+carriers+for+drug+delivery+systems.">Polysaccharide-based aerogels-Promising biodegradable carriers for drug delivery systems.</a> Carbohydr. Polym. 86 (4), 1425-1438 (2011).</li><li>Alnaief, M., Alzaitoun, M. A., Garc&#237;a-Gonz&#225;lez, C. A., Smirnova, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+biodegradable+nanoporous+microspherical+aerogel+based+on+alginate.">Preparation of biodegradable nanoporous microspherical aerogel based on alginate.</a> Carbohydr. Polym. 84 (3), 1011-1018 (2011).</li><li>Gurikov, P., Raman, S. P., Weinrich, D., Fricke, M., Smirnova, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+to+alginate+aerogels:+carbon+dioxide+induced+gelation.">A novel approach to alginate aerogels: carbon dioxide induced gelation.</a> RSC Adv. 5, 7812-7818 (2015).</li><li>Meyssami, B., Balaban, M. O., Teixeira, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+pH+in+model+systems+pressurized+with+carbon+dioxide.">Prediction of pH in model systems pressurized with carbon dioxide.</a> Biotechnol. Prog. 8 (2), 149-154 (1992).</li><li>Subrahmanyam, R., Gurikov, P., Dieringer, P., Sun, M., Smirnova, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+Road+to+Biopolymer+Aerogels-Dealing+with+the+Solvent.">On the Road to Biopolymer Aerogels-Dealing with the Solvent.</a> Gels. 1 (2), 291-313 (2015).</li><li>Garc&#237;a-Gonz&#225;lez, C. A., Camino-Rey, M. C., Alnaief, M., Zetzl, C., Smirnova, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supercritical+drying+of+aerogels+using+CO2:+Effect+of+extraction+time+on+the+end+material+textural+properties.">Supercritical drying of aerogels using CO2: Effect of extraction time on the end material textural properties.</a> J. Supercrit. Fluids. 66, 297-306 (2012).</li><li>&#214;zbak&#305;r, Y., Erkey, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+and+theoretical+investigation+of+supercritical+drying+of+silica+alcogels.">Experimental and theoretical investigation of supercritical drying of silica alcogels.</a> J. Supercrit. Fluids. 98, 153-166 (2015).</li><li>Rudaz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aeropectin:+Fully+Biomass-Based+Mechanically+Strong+and+Thermal+Superinsulating+Aerogel.">Aeropectin: Fully Biomass-Based Mechanically Strong and Thermal Superinsulating Aerogel.</a> Biomacromolecules. 15 (6), 2188-2195 (2014).</li><li>Quraishi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+non-cytotoxic+alginate-lignin+hybrid+aerogels+as+scaffolds+for+tissue+engineering.">Novel non-cytotoxic alginate-lignin hybrid aerogels as scaffolds for tissue engineering.</a> J. Supercrit. Fluids. 105, 1-8 (2015).</li><li>Martins, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+macroporous+alginate-based+aerogels+for+biomedical+applications.">Preparation of macroporous alginate-based aerogels for biomedical applications.</a> J. Supercrit. Fluids. 106, 152-159 (2015).</li><li>Davidovich-Pinhas, M., Bianco-Peled, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+analysis+of+alginate+swelling.">A quantitative analysis of alginate swelling.</a> Carbohydr. Polym. 79 (4), 1020-1027 (2010).</li><li>Mallepally, R. R., Bernard, I., Marin, M. A., Ward, K. R., McHugh, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superabsorbent+alginate+aerogels.">Superabsorbent alginate aerogels.</a> J. Supercrit. Fluids. 79, 202-208 (2013).</li><li>Porta, G. D., Del Gaudio, P., De Cicco, F., Aquino, R. P., Reverchon, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supercritical+Drying+of+Alginate+Beads+for+the+Development+of+Aerogel+Biomaterials:+Optimization+of+Process+Parameters+and+Exchange.">Supercritical Drying of Alginate Beads for the Development of Aerogel Biomaterials: Optimization of Process Parameters and Exchange.</a> Ind. Eng. Chem. Res. 52 (34), 12003-12009 (2013).</li><li>Brown, Z. K., Fryer, P. J., Norton, I. T., Bridson, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drying+of+agar+gels+using+supercritical+carbon+dioxide.">Drying of agar gels using supercritical carbon dioxide.</a> J. Supercrit. Fluids. 54 (1), 89-95 (2010).</li><li>Betz, M., Garc&#237;a-Gonz&#225;lez, C. A., Subrahmanyam, R. P., Smirnova, I., Kulozik, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+novel+whey+protein-based+aerogels+as+drug+carriers+for+life+science+applications.">Preparation of novel whey protein-based aerogels as drug carriers for life science applications.</a> J. Supercrit. Fluids. 72, 111-119 (2012).</li><li>Antonyuk, S., Heinrich, S., Gurikov, P., Raman, S., Smirnova, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+coating+and+wetting+on+the+mechanical+behaviour+of+highly+porous+cylindrical+aerogel+particles.">Influence of coating and wetting on the mechanical behaviour of highly porous cylindrical aerogel particles.</a> Powder Technol. 285, 34-43 (2015).</li></ol>

The authors declare that they have no competing financial interests.

By using the CO2 induced gelation technique, one can eliminate the need for chemical substitutes (for example acetic acid or glucono delta-lactone (GDL)) required for inducing the crosslinking of the biopolymer. The surface areas of the amidated pectin aerogels are in the higher ranges of literature values5, however the pore volumes are much higher than those presented in literature5. Higher pore volumes were also observed for alginate aerogels prepared by CO2 induced gelation7. However, it remains to be verified whether the reason for this high pore volumes (4-150 nm pore size range) is due to the gelation technique or an inherent property of the biopolymers previously not addressed in literature. Pectin aerogels have been reported in literature to possess superinsulating properties12 and alginate aerogels prepared by this technique also possess thermal conductivities in the superinsulating range3,7. Therefore, the amidated pectin aerogels produced by this technique may also be envisaged to possess superinsulating properties.
The rate of depressurization in Protocol Section 2 is an important step in the hydrogel preparation. Fast depressurization can lead to increased macroporosity of the gels. This phenomena can be applied for tissue engineering applications where macroporosity of the material with interconnectivity is an important feature for the growth and proliferation of cells13,14. In addition, the crosslinking degree in Protocol Section 1 plays an important role in the syneresis and swelling property of the amidated pectin hydrogels. This is similar to alginate hydrogels whose swelling behavior is influenced by the crosslinker concentration as well15. Thereby aerogels made by amidated pectin can also be tuned to possess superabsorbent property similar to those reported for alginate aerogels16.
By using the CO2 induced gelation considering amidated pectin (or alginate) as the primary system, further diversity can be incorporated into the aerogels by introducing different cross linking agents and biopolymer combinations. Several metal carbonates (e.g., zinc, nickel, cobalt, copper, strontium, barium) could be used for cross linking3, where cations can be released by pH lowering in aqueous media with pressurized CO2 (3-5 MPa). However, insoluble salts of some of these cations may not form stable dispersions for lower biopolymer concentrations and can settle down at the bottom leading to inhomogeneous gels. This is a general issue with the internal setting gelation method including CO2 induced gelation3 and thereby, the technique&#39;s usability for an application should be evaluated on a case to case basis.
Various blends prepared using water soluble biopolymers such as starch, carrageenan, methyl and carboxy methyl cellulose, gellan gum, lignin, gelatin and others; water soluble synthetic polymers such as polyethylene glycol (PEG), polyvinyl alcohol (PVA), Pluronic P-123 and others; and water soluble inorganic precursors such as sodium silicate can be also mixed with amidated pectin to produce hybrid aerogels similar to alginate2 with tunable properties.
As supercritical CO2 drying (scCO2 drying) is a quintessential step in aerogel production, any combination of pre-processing steps such as solvent exchange and drying using CO217,18 or gelation, solvent exchange and drying using CO27 could provide a clear processing advantage. The advantage is envisioned as integrated one pot process: where biopolymer dispersions can be converted into biopolymer aerogels using CO2 as the main processing medium in a single autoclave. For certain pharmaceutical applications, one can also envision performing a four step: gelation, solvent exchange, supercritical drying and active component loading5,19 process in a single autoclave using CO2 as the processing medium. Post treatment such as protective coating of drug loaded aerogels in certain cases is necessary for targeted drug release20.
To conclude, the present work demonstrates the use of pressurized CO2 for gelation of amidated pectin based systems. In addition, the use of pressurized CO2 as a common medium for precursor to product conversion for target applications in a single autoclave is envisaged.

Aerogels are a class of porous materials that can be prepared using a variety of precursors ranging from inorganic (such as silica, titania, zirconia and others), synthetic (such as resorcinol formaldehyde, polyurethane and others) or biopolymers (polysaccharides, proteins and others)2. What sets them apart from the conventional porous materials is their ability to simultaneously possess all the three characteristics; namely high surface area, ultra-low density and mesoporous pore size distribution (i.e., pore sizes from 2-50 nm). With aforementioned characteristics, aerogels are extensively applied in the fields of insulation, biomedicine, catalysis, adsorption and absorption applications, pharmaceuticals and neutraceuticals2. Taking into account the above possibilities, the production of biopolymer gel systems and their subsequent transformation to aerogels opens up a multitude of opportunities towards high value added bio based materials. Such an endeavor is taken up in this study using amidated pectin as an example.
Aerogels are typically produced by the sol-gel technique. Gels are systems consisting of liquid entrapped in a matrix and can be prepared by covalent, ionic, pH induced, thermal or cryo cross linking3. For this specific system, we utilize ionic crosslinking; i.e., a bivalent cation (e.g., calcium) to crosslink biopolymeric chains together. To perform controllable ionic crosslinking of biopolymers such as amidated pectin or alginate, one can utilize the diffusion method or the internal setting method4. In the diffusion method, gelation occurs at first in the outer layer followed by diffusion propagation, as the cations diffuse from outer solution into an amidated pectin or alginate droplet or layer4. In the internal setting method, the insoluble form of the crosslinker is homogenously dispersed in the biopolymer solution and cations are released by initiating a pH change4,5,6. However, both techniques face an issue regarding the homogeneity of the final gel when produced in slab or monolithic form. This work demonstrates the use of high pressure CO2 (5 MPa) for the production of amidated pectin hydrogels building further on previous works on alginate gels3,7. In brief, it is an internal setting gelation technique that utilizes pressurized CO2 to reduce pH instead of weak acids to produce homogeneous gels. With an increase in the pressure, the solubility of carbon dioxide in water increases accompanied by a lowering of pH to 3.08. This causes calcium carbonate to solubilize, releasing the calcium ions. The calcium ions crosslink with the amidated pectin biopolymer to yield hydrogels. Stable homogeneous gels down to very low biopolymer concentrations (0.05 wt%) could be produced using this technique7.
As gelation takes place in an aqueous medium, solvent exchange to an organic solvent is required due to a miscibility gap in the CO2/water system. Typically low molecular weight alcohols (methanol/ethanol/isopropanol) and ketones (acetone) can be used for the solvent exchange process. However, direct soaking in a bath with pure ethanol or other organic solvents leads to significant irreversible shrinkage. To avoid this drawback, stepwise solvent exchange is performed5,9. When the solvent concentration inside the gel reaches &#62;98%, the organic solvent is dried with supercritical CO2 (12 MPa) leaving behind an aerogel.

<table border="0" cellpadding="0" cellspacing="0" width="1321">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:289px;">Equipment</td>
			<td style="width:196px;"></td>
			<td style="width:240px;"></td>
			<td style="width:596px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultraturrax homogenizer</td>
			<td>IKA, Germany</td>
			<td>T 25 Digital</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polypropylene molds</td>
			<td>TH. Geyer, Germany</td>
			<td>9,033,201</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High pressure autoclave&#160;</td>
			<td>Ernst Haage, Germany</td>
			<td>custom made</td>
			<td>Setup constuction done in-house</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Compressor</td>
			<td>Andreas Hofer</td>
			<td>MKZ 185-40</td>
			<td>Setup constuction done in-house</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nitrogen adsorption</td>
			<td>Quantachrome</td>
			<td>Nova 4000e</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Density meter</td>
			<td>Anton Paar</td>
			<td>DMA 4000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Chemicals</td>
			<td style="width:196px;"></td>
			<td style="width:240px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amidated pectin</td>
			<td>Herbstreith and Fox, Germany</td>
			<td>CU 025</td>
			<td>CAS # 56645-02-4; provided by company for research purposes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Hydroxide</td>
			<td>Sigma Aldrich, Germany</td>
			<td style="width:240px;">S8045</td>
			<td>CAS # 1310-73-2; required only in case the pectin solution needs to be neutralized to pH 6.5-7.5</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Calcium carbonate</td>
			<td>Magnesia GmbH, Germany</td>
			<td style="width:240px;">4421 Calcium carbonat, leicht, pr&#228;zipitiert, EP, E170</td>
			<td>CAS # 471-34-1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol, 99.8 %</td>
			<td>Sigma Aldrich, Germany</td>
			<td>32205</td>
			<td>CAS # 64-17-5</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carbon dioxide, 99.9 %</td>
			<td>AGA Gas GmbH, Germany</td>
			<td></td>
			<td>CAS # 124-38-9; in-house tank available (3 ton)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Deionised Water</td>
			<td></td>
			<td></td>
			<td>CAS # 7732-18-5;&#160; available in-house (6.4-7.0 pH)</td>
		</tr>
	</tbody>
</table>

preparation of biopolymer aerogels using green solvents

Although the first reports on aerogels made by Kistler1 in the 1930s dealt with aerogels from both inorganic oxides (silica and others) and biopolymers (gelatin, agar, cellulose), only recently have biomasses been recognized as an abundant source of chemically diverse macromolecules for functional aerogel materials. Biopolymer aerogels (pectin, alginate, chitosan, cellulose, etc.) exhibit both specific inheritable functions of starting biopolymers and distinctive features of aerogels (80-99% porosity and specific surface up to 800 m2/g). This synergy of properties makes biopolymer aerogels promising candidates for a wide gamut of applications such as thermal insulation, tissue engineering and regenerative medicine, drug delivery systems, functional foods, catalysts, adsorbents and sensors. This work demonstrates the use of pressurized carbon dioxide (5 MPa) for the ionic cross linking of amidated pectin into hydrogels. Initially a biopolymer/salt dispersion is prepared in water. Under pressurized CO2 conditions, the pH of the biopolymer solution is lowered to 3 which releases the crosslinking cations from the salt to bind with the biopolymer yielding hydrogels. Solvent exchange to ethanol and further supercritical CO2 drying (10 - 12 MPa) yield aerogels. Obtained aerogels are ultra-porous with low density (as low as 0.02 g/cm3), high specific surface area (350 - 500 m2/g) and pore volume (3 - 7 cm3/g for pore sizes less than 150 nm).

The typical hydrogels obtained after gelation step with higher crosslinking degree (q=2) (as instructed in Protocol section 2) are shown in Figure 1. The samples on the left (sample A and B) are the 2 wt% and 1 wt% pectin gels obtained by CO2 induced gelation. By decreasing the biopolymer concentration (0.5 wt% or lower), the gels become transparent (sample C). Further reduction in the biopolymer concentration (0.25 wt%) also yields stable hydrogels (sample D) but these gels are very fragile and can break when handling. The bubbles observed inside the hydrogels are created during depressurization when the dissolved CO2 leaves the gel water system due to decrease in CO2 solubility.
The amidated pectin aerogel characteristics are presented in the Table 1. The obtained aerogels are ultra-porous with low density (as low as 0.013 g/cm3) measured as the ratio between the mass of the aerogel and its volume. The surface area is measured by the nitrogen adsorption. For pectin aerogels, it yielded a specific surface area between 350 - 500 m2/g. The pore volume for pore sizes in the 4-150 nm range is measured by the Kelvin model of pore filling using nitrogen (BJH method). The pore volume for the amidated pectin aerogels was between 3 - 7 cm3/g for pore sizes between 4 and 150 nm.
<img alt="Figure 1" src="/files/ftp_upload/54116/54116fig1.jpg" /> 
Figure 1. Amidated pectin hydrogels with higher crosslinking degree (q=2). Top left: 2 wt% (Sample A); top right: 1 wt% (Sample B); bottom left: 0.5 wt% (Sample C); bottom right: 0.25 wt% (Sample D). Gels become transparent with decreasing biopolymer concentration. The bubbles are produced during CO2 depressurization. <a href="https://www.jove.com/files/ftp_upload/54116/54116fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Pectin concentration [wt%]</td>
			<td>Cross-linking degree q</td>
			<td>Bulk density [g/cm3]</td>
			<td>Specific surface area [m2/g]</td>
			<td>Specific pore volume [cm3/g]</td>
			<td>Average pore size (diameter) [nm]</td>
		</tr>
		<tr>
			<td>2.00</td>
			<td>1</td>
			<td>0.081</td>
			<td>502</td>
			<td>4.1</td>
			<td>14</td>
		</tr>
		<tr>
			<td>1.00</td>
			<td>1</td>
			<td>0.044</td>
			<td>491</td>
			<td>7.1</td>
			<td>27</td>
		</tr>
		<tr>
			<td>0.50</td>
			<td>1</td>
			<td>0.035</td>
			<td>357</td>
			<td>3.8</td>
			<td>27</td>
		</tr>
		<tr>
			<td>0.25</td>
			<td>1</td>
			<td>0.013</td>
			<td>335</td>
			<td>4.9</td>
			<td>41</td>
		</tr>
		<tr>
			<td>2.00</td>
			<td>2</td>
			<td>0.069</td>
			<td>447</td>
			<td>3.1</td>
			<td>13</td>
		</tr>
		<tr>
			<td>1.00</td>
			<td>2</td>
			<td>0.048</td>
			<td>441</td>
			<td>3.6</td>
			<td>26</td>
		</tr>
		<tr>
			<td>0.50</td>
			<td>2</td>
			<td>0.030</td>
			<td>429</td>
			<td>5.8</td>
			<td>25</td>
		</tr>
		<tr>
			<td>0.25</td>
			<td>2</td>
			<td>0.017</td>
			<td>347</td>
			<td>5.0</td>
			<td>24</td>
		</tr>
	</tbody>
</table>
Table 1. Characteristics of the amidated pectin aerogels.

Watch this Scientific Journal Video about Preparation of Biopolymer Aerogels Using Green Solvents at JoVE.com

Preparation of Biopolymer Aerogels Using Green Solvents

A new way for the production of biopolymer-based aerogels by carbon dioxide (CO2) induced gelation is shown. The technique utilizes pressurized carbon dioxide (5 MPa) for the production of biopolymer hydrogels and supercritical CO2 (12 MPa) to convert gels into aerogels. The only solvents needed besides CO2 are water and ethanol.

Although the first reports on aerogels made by Kistler1 in the 1930s dealt with aerogels from both inorganic oxides (silica and others) and biopolymers ...

preparation-of-biopolymer-aerogels-using-green-solvents

Universidad San Sebastian

Research

JoVE Journal

Chemistry

17.5K Views.  Hamburg University of Technology. A new way for the production of biopolymer-based aerogels by carbon dioxide (CO2) induced gelation is shown. The technique utilizes pressurized carbon dioxide (5 MPa) for the production of biopolymer hydrogels and supercritical CO2 (12 MPa) to convert gels into aerogels. The only solvents needed besides CO2 are water and ethanol.

Video: Preparation of Biopolymer Aerogels Using Green Solvents

Exploring the Radical Nature of a Carbon Surface by Electron Paramagnetic Resonance and a Calibrated Gas Flow

While the first Electron Paramagnetic Resonance (EPR) studies regarding the effects of oxidation on the structure and stability of carbon radicals date back to the early 1980s the focus of these early papers primarily characterized the changes to the structures under extremely harsh conditions (pH or temperature)1-3. It is also known that paramagnetic molecular oxygen undergoes a Heisenberg spin exchange interaction with stable radicals that extremely broadens the EPR signal4-6. Recently, we reported interesting results where this interaction of molecular oxygen with a certain part of the existing stable radical structure can be reversibly affected simply by flowing a diamagnetic gas through the carbon samples at STP7. As flows of He, CO2, and N2 had a similar effect these interactions occur at the surface area of the macropore system.
This manuscript highlights the experimental techniques, work-up, and analysis towards affecting the existing stable radical nature in the carbon structures. It is hoped that it will help towards further development and understanding of these interactions in the community at large.

Stable radicals that are present in carbon substrates interact with paramagnetic oxygen through a Heisenberg spin exchange. This interaction can be significantly reduced under STP conditions by flowing a diamagnetic gas over the carbon system. This manuscript describes a simple method to characterize the nature of those radicals.

While the first Electron Paramagnetic Resonance (EPR) studies regarding the effects of oxidation on the structure and stability of carbon radicals date ...

Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible to ensure consistent product quality over time using this technique. We here show how to interface a commercially available flow unit with a Raman spectrometer. The Raman flow cell is placed after the back-pressure regulator, meaning that it can be operated at atmospheric pressure. In addition, the fact that the product stream passes through a length of tubing before entering the flow cell means that the material is at RT. It is important that the spectra are acquired under isothermal conditions since Raman signal intensity is temperature dependent. Having assembled the apparatus, we then show how to monitor a chemical reaction, the piperidine-catalyzed synthesis of 3-acetylcoumarin from salicylaldehyde and ethyl acetoacetate being used as an example. The reaction can be performed over a range of flow rates and temperatures, the in-situ monitoring tool being used to optimize conditions simply and easily.

Real-time monitoring allows for fast optimization of reactions performed using continuous-flow processing. Here the preparation of 3-acetylcoumarin is used as an example. The apparatus for performing in-situ Raman monitoring is described, as are the steps required to optimize the reaction.

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible ...

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. The most common techniques utilized are imaging modalities such as Magnetic Resonance Imaging (MRI), Positron Emission Topography (PET), and Computed Topography (CT) and are optimal for understanding the physical structure of the disease but can only be performed once every four to six weeks due to the use of imaging agents and overall cost. With this in mind, the development of &#8220;point of care&#8221; techniques, such as biosensors, which evaluate the stage of disease and/or efficacy of treatment in the clinician&#8217;s office and do so in a timely manner, would revolutionize treatment protocols.1 As a means to exploring ferrocene based biosensors for the detection of biologically relevant molecules2, methods were developed to produce ferrocene-biotin bio-conjugates described herein. This report will focus on a biotin-ferrocene-cysteine system that can be immobilized on a gold surface.

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids (BPCA)

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as char and soot. PyC is ubiquitous in the environment due to its long persistence, and its abundance might even increase with the projected increase in global wildfire activity and the continued burning of fossil fuel. PyC is also increasingly produced from the industrial pyrolysis of organic wastes, which yields charred soil amendments (biochar). Moreover, the emergence of nanotechnology may also result in the release of PyC-like compounds to the environment. It is thus a high priority to reliably detect, characterize and quantify these charred materials in order to investigate their environmental properties and to understand their role in the carbon cycle.
Here, we present the benzene polycarboxylic acid (BPCA) method, which allows the simultaneous assessment of PyC&#39;s characteristics, quantity and isotopic composition (13C and 14C) on a molecular level. The method is applicable to a very wide range of environmental sample materials and detects PyC over a broad range of the combustion continuum, i.e., it is sensitive to slightly charred biomass as well as high temperature chars and soot. The BPCA protocol presented here is simple to employ, highly reproducible, as well as easily extendable and modifiable to specific requirements. It thus provides a versatile tool for the investigation of PyC in various disciplines, ranging from archeology and environmental forensics to biochar and carbon cycling research.

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids BPCA

We present the benzene polycarboxylic acid (BPCA) method for assessing pyrogenic carbon (PyC) in the environment. The compound-specific approach uniquely provides simultaneous information about the characteristics, quantity and isotopic composition (13C and 14C) of PyC.

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as ...

Green and Low-cost Production of Thermally Stable and Carboxylated Cellulose Nanocrystals and Nanofibrils Using Highly Recyclable Dicarboxylic Acids

Here we demonstrate potentially low cost and green productions of high thermally stable and carboxylated cellulose nanocrystals (CNCs) and nanofibrils (CNF) from bleached eucalyptus pulp (BEP) and unbleached mixed hardwood kraft pulp (UMHP) fibers using highly recyclable dicarboxylic solid acids. Typical operating conditions were acid concentrations of 50 - 70 wt% at 100 &#176;C for 60 min and 120 &#176;C (no boiling at atmospheric pressure) for 120 min, for BEP and UMHP, respectively. The resultant CNCs have a higher thermal degradation temperature than their corresponding feed fibers and carboxylic acid group content from 0.2 - 0.4 mmol/g. The low strength (high pKa of 1.0 - 3.0) of organic acids also resulted in CNCs with both longer lengths of approximately 239 - 336 nm and higher crystallinity than CNCs produced using mineral acids. Cellulose loss to sugar was minimal. Fibrous cellulosic solid residue (FCSR) from the dicarboxylic acid hydrolysis was used to produce carboxylated CNFs through subsequent mechanical fibrillation with low energy input.

Here we demonstrate a novel method for green and sustainable productions of highly thermally stable and carboxylated cellulose nanocrystals (CNC) and nanofibrils (CNF) using highly recyclable solid dicarboxylic acids.

Here we demonstrate potentially low cost and green productions of high thermally stable and carboxylated cellulose nanocrystals (CNCs) and nanofibrils ...

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the inverse electron demand Diels-Alder (IEDDA) cycloaddition between a radiolabeled tetrazines and trans-cyclooctene (TCO) linked to a biomolecule has proven to be a highly effective bioorthogonal approach to imaging specific biological targets. Despite the fact that technetium-99m remains the most widely used isotope in diagnostic nuclear medicine, there is a scarcity of methods for preparing 99mTc-labeled tetrazines. Herein we report the preparation of a family of tridentate-chelate-tetrazine derivatives and their Tc(I) complexes. These hitherto unknown compounds were radiolabeled with 99mTc using a microwave-assisted method in 31% to 83% radiochemical yield. The products are stable in saline and PBS and react rapidly with TCO derivatives in vitro. Their in vivo pre-targeting abilities were demonstrated using a TCO-bisphosphonate (TCO-BP) derivative that localizes to regions of active bone metabolism or injury. In murine studies, the 99mTc-tetrazines showed high activity concentrations in knees and shoulder joints, which was not observed when experiments were performed in the absence of TCO-BP. The overall uptake in non-target organs and pharmacokinetics varied greatly depending on the nature of the linker and polarity of the chelate.

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...

Fabrication and Testing of Catalytic Aerogels Prepared Via Rapid Supercritical Extraction

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three preparation methods are described: (a) the incorporation of metal salts into silica or alumina wet gels using an impregnation method; (b) the incorporation of metal salts into alumina wet gels using a co-precursor method; and (c) the addition of metal nanoparticles directly into a silica aerogel precursor mixture. The methods utilize a hydraulic hot press, which allows for rapid (&#60;6 h) supercritical extraction and results in aerogels of low density (0.10 g/mL) and high surface area (200-800 m2/g). While the work presented here focuses on the use of copper salts and copper nanoparticles, the approach can be implemented using other metal salts and nanoparticles. A protocol for testing the three-way catalytic ability of these aerogels for automotive pollution mitigation is also presented. This technique uses custom-built equipment, the Union Catalytic Testbed (UCAT), in which a simulated exhaust mixture is passed over an aerogel sample at a controlled temperature and flow rate. The system is capable of measuring the ability of the catalytic aerogels, under both oxidizing and reducing conditions, to convert CO, NO and unburned hydrocarbons (HCs) to less harmful species (CO2, H2O and N2). Example catalytic results are presented for the aerogels described.

Here we present protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms. Methods for preparing materials using copper salts and copper-containing nanoparticles are featured. Catalytic testing protocols demonstrate the effectiveness of these aerogels for three-way catalysis applications.

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three ...

Preparation of Functional Silica Using a Bioinspired Method

The goal of the protocols described herein is to synthesize bioinspired silica materials, perform enzyme encapsulation therein, and partially or totally purify the same by acid elution. By combining sodium silicate with a polyfunctional bioinspired additive, silica is rapidly formed at ambient conditions upon neutralization. 
The effect of neutralization rate and biomolecule addition point on silica yield are investigated, and biomolecule immobilization efficiency is reported for varying addition point. In contrast to other porous silica synthesis methods, it is shown that the mild conditions required for bioinspired silica synthesis are fully compatible with the encapsulation of delicate biomolecules. Additionally, mild conditions are used across all synthesis and modification steps, making bioinspired silica a promising target for the scale-up and commercialization as both a bare material and active support medium.
The synthesis is shown to be highly sensitive to conditions, i.e., the neutralization rate and final synthesis pH, however tight control over these parameters is demonstrated through the use of auto titration methods, leading to high reproducibility in reaction progression pathway and yield. 
Therefore, bioinspired silica is an excellent active material support choice, showing versatility towards many current applications, not limited to those demonstrated here, and potency in future applications.

Here, we present a protocol to synthesize bioinspired silica materials and immobilize enzymes therein. Silica is synthesized by combining sodium silicate and an amine &#39;additive&#39;, which neutralize at a controlled rate. Material properties and function can be altered either by in situ enzyme immobilization or post-synthetic acid elution of encapsulated additives.

The goal of the protocols described herein is to synthesize bioinspired silica materials, perform enzyme encapsulation therein, and partially or totally ...

A Rapid Synthesis Method for Au, Pd, and Pt Aerogels Via Direct Solution-Based Reduction

Here, a method to synthesize gold, palladium, and platinum aerogels via a rapid, direct solution-based reduction is presented. The combination of various precursor noble metal ions with reducing agents in a 1:1 (v/v) ratio results in the formation of metal gels within seconds to minutes compared to much longer synthesis times for other techniques such as sol-gel. Conducting the reduction step in a microcentrifuge tube or small volume conical tube facilitates a proposed nucleation, growth, densification, fusion, equilibration model for gel formation, with final gel geometry smaller than the initial reaction volume. This method takes advantage of the vigorous hydrogen gas evolution as a by-product of the reduction step, and as a consequence of reagent concentrations. The solvent accessible specific surface area is determined with both electrochemical impedance spectroscopy and cyclic voltammetry. After rinsing and freeze drying, the resulting aerogel structure is examined with scanning electron microscopy, X-ray diffractometry, and nitrogen gas adsorption. The synthesis method and characterization techniques result in a close correspondence of aerogel ligament sizes. This synthesis method for noble metal aerogels demonstrates that high specific surface area monoliths may be achieved with a rapid and direct reduction approach.

A rapid, direct solution-based reduction synthesis method to obtain Au, Pd, and Pt aerogels is presented.

Here, a method to synthesize gold, palladium, and platinum aerogels via a rapid, direct solution-based reduction is presented. The combination of various ...

Achieving Moderate Pressures in Sealed Vessels Using Dry Ice As a Solid CO2 Source

Herein is presented a general strategy to perform reactions under mild to moderate CO2 pressures with dry ice. This technique obviates the need for specialized equipment to achieve modest pressures, and can even be used to achieve higher pressures in more specialized equipment and sturdier reaction vessels. At the end of the reaction, the vials can be easily depressurized by opening at room temperature. In the present example CO2 serves as both a putative directing group as well as a way to passivate amine substrates, thereby preventing oxidation during the organometallic reaction. In addition to being easily added, the directing group is also removed under vacuum, obviating the need for extensive purification to remove the directing group. This strategy allows the facile &#947;-C(sp3)-H arylation of aliphatic amines and has the potential to be applied to a variety of other amine-based reactions.

Achieving Moderate Pressures in Sealed Vessels Using Dry Ice As a Solid CO2 Source

Here we present a protocol for performing reactions in simple reaction vessels under low-to-moderate pressures of CO2. The reactions can be performed in a variety of vessels simply by administering the carbon dioxide in the form of dry ice, without the need for costly or elaborate equipment or set-ups.

Herein is presented a general strategy to perform reactions under mild to moderate CO2 pressures with dry ice. This technique obviates the need for ...