Ezchrom elite 3

Average molecular weight distribution of chitosan (50 kDa) and fatty acid grafted chitosan polymers was determined using size exclusion high performance liquid chromatography (SEC-HPLC).¹¹ Samples were prepared at a concentration of 10 mg/mL in 1% acetic acid (v/v) in deionized water. An Ultrahydrogel 250 (Waters, MA, U.S.) column was used in combination with the Agilent 1120 Compact LC system coupled with an Agilent 1200 series refractive index (RI) detector (Agilent, DE, U.S.) set at 35 °C, with 1% (v/v) acetic acid in deionized water as mobile phase, 0.5 mL/min flow rate, and 60 min run time. Data were analyzed using EZChrom Elite 3.3.2 software (Agilent, CA, U.S.).

Insulin released from optimized formulation in vitro was further investigated for thermal stability using nano DSC, chemical stability using RP-HPLC, and conformational stability using CD spectroscopy. Nano DSC was used to check the thermal transition midpoint temperature and enthalpy change of released insulin as mentioned earlier. Chemical stability was determined by RP-HPLC using Agilent 1120 compact LC system using chromatographic conditions presented in table 2. Data acquisition and analysis was performed using EZChrom Elite™ 3.3.2 software (Agilent, CA, USA). CD spectroscopy was used check the tertiary and secondary structural stability by observing the sample spectrum in near-UV region (250 – 300 nm) and far-UV region (200 – 250 nm), respectively. Scans were performed at a rate of 5 nm/min at 20 °C using a 0.1 cm path length quartz cuvette. Fresh PBS scan was subtracted from the sample scan prior to data analysis to remove any background interference. Spectra manager®2 software provided with the instrument was used for analysis of spectrum and secondary structure estimation.