(d) Magnification from the ice nucleation region coming from Panel a. upon warming of partly glassy or entirely glassy mother CA/H2O. The formation of two FCS1and FCS22regions during the freezing upon warming to our best knowledge has never been reported before. Using an optical cryo-microscope, we are able to observe the formation of a continuous ice framework (IF) as well as morphology and reciprocal distribution of IF/(FCS1+ FCS2). Our results Optovin give a new look at the freezing and glass transition behavior of aqueous solutions and can be used for the optimization of lyophilization and very cold of foods and biopharmaceutical formulations, among many other applications where very cold plays a crucial role. == 1 . Launch == Citric acid (CA), C6H8O7, is actually a weak organic acid that is naturally encountered in a variety of fruits and vegetables, especially lemons and limes, and is commercially produced in very large amount by microbial fermentation of carbohydrates. The usual form of CA is usually CA-monohydrate, C6H8O7H2O, which is crystallized by the slower evaporation of water coming from cold saturated solutions, whereas anhydrous CA is crystallized from sizzling saturated answer. 1CA by virtue of its hydroxyl OH and carboxyl COOH groups is capable to form hydrogen bonding between CA molecules themselves and with solvent H2O molecules, 2, 3which results Optovin in a variety of unique properties of CA/H2O solutions. Strong hydrogen bonding is believed to be a property that may prevent the crystallization of CA from cooled melt/solutions and be responsible for a liquidglass transition4, 5that is important for freeze-drying (lyophilization) and freezing of pharmaceutical formulations. CA is usually widely used in foods and beverages as a flavoring and preservative ingredient, 5, 6industry, 68pharmaceutics because excipient9for freeze-dried formulations, 10and in order to produce amorphous multicomponent excipients having high cup transition heat, Tg, which could reduce or prevent protein denaturation in lyophilized formulations. 11, 12CA increases solubility of poorly water-soluble drugs13, 14and efficiently maintains pH in the range from 3 to 6. 2 that increases the stability of therapeutic proteins in frozen large scale formulations during storage. 15, 16CA is also used for the solubilization and sustained delivery of anti-HIV (human immunodeficiency virus) drug17and in cells engineering (synthesis of biodegradable scaffold), 1822biochemistry, etc . Although CA is usually widely used in the different fields of technology, industry, and low temperature digesting, there are still gaps in understanding of freeze-induced phase separation into pure ice and freeze-concentrated solution (FCS), glass transition behavior of formed FCS, and the morphology and reciprocal distribution of ice and glassy FCS in freezing solutions. In this post, we present the differential scanning calorimetry (DSC) and optical cryo-microscope (OC-M) results of the research of 1064 wt % CA solutions subjected to diverse cooling and warming rates. Our results obtained from the combined DSC and OC-M measurements give a clear picture of how ice and FCSs of different concentrations are created during Mouse monoclonal to CD4/CD8 (FITC/PE) the very cold upon cooling and subsequent warming and reveal a variety of liquidglass and reverse glassliquid transitions, which have not been observed before. == 2 . Experimental Section == We prepared 1064 wt % CA solutions by mixing > 99% anhydrous citric acid (Merck) with the corresponding amount of ultrapure water. The solutions of focus larger than the solubility limit of 62 wt % CA at 295 K23, 24were prepared by slow heating system. We analyzed the phase transformations and glass transitions of CA/H2O using a Mettler Toledo DSC 822 calorimeter at the checking cooling/warming price of 3, 0. 5, and 0. 1 K/min in the temperature region between 320 and 133 K. Such cooling rates are usually applied during the lyophilization of large level food and biopharmaceutical formulations. For the DSC measurements, we packed and then chilly sealed a half-sphere answer drop in an aluminum (Al) crucible of 40 L by volume. The mass and diameter of drops were 56 mg and 1 . five mm, respectively. We also performed measurements at three or more K/min upon warming of quenched CA/H2O drops. To this end we placed an Al crucible with CA/H2O drop into liquid N2and then immediately inserted the crucible into the precooled calorimeter. In Optovin this procedure, the cooling rate is usually estimated to be 1001000 K/s. DSC calibration and details about measurements are described elsewhere. 25, 26To verify the reproducibility of results, we performed a number of repeated DSC measurements, which were of two types: (i) we sometimes.