Actually, more water molecules are probably associated with the hydronium ion, increasing its sphere of hydration or effective diameter with a hydrated ionic formula of H 9O 4 + (( 7). The hydrogen ion (H +) is commonly denoted as associating with one water molecule and is thus referred to as the hydronium ion (H 3O +). A and B are constants with values of 0.5085 and 0.3281 at 25 ☌. Such hydrated radii are predicted by the Debye-Hückel Equation (Equation 1), in which ? is the activity coefficient, z is the charge of the ion, is the ionic strength of the aqueous solution, and a is the effective diameter. When salts are dissolved in water and become ionized, spheres of hydration are formed around their positively and negatively charged ions with radii inversely proportional to the ions’ nonhydrated radii and directly proportional to their charge (( 5, 6). Although that is true for solids to a great extent, aqueous solutions with salts represent a special case. The Inorganics Question: NIR spectroscopists often say that inorganic compounds are ill suited for NIR analysis because they lack the required change in dipole moment. Sodium hydroxide solution, which we used is this study, is another common choice. The pH is usually controlled by addition of sodium bicarbonate and CO 2 (( 4). Most mammalian cells thrive in the 7.0–7.5 pH range, and it is important to keep their culture medium buffered at that optimum level. Cell lines thrive in different pH ranges, which may also depend upon the formulation of their cell culture medium. Pure water at pH 7.0 has an equal number of hydroxyl (OH –) ions as hydronium ions and is thus neither acidic nor basic.ĭuring metabolism, mammalian cells produce lactic and carbonic acids that tend to drive the pH of their culture below 7.0. A pH of 7.0 means that there are 10 -7 mol/L of hydronium ions present. The pH value is logarithmic shorthand for specifying the number of hydrogen (H +) or hydronium (H 3O +) ions per liter of a solution. Osmolality measurement is performed to determine the concentration of inorganic salts present in a solution, ensuring proper cell viability and optimal cell membrane transport in cultures. It is independent of both temperature and the amount of solute added (unlike osmolarity) because it is related to unit mass rather than unit volume. Osmolality measures the number of moles per kilogram of substances that cause osmotic pressure in cell culture media. OH, CH, NH, and SH bonds have the strongest overtone absorbances in the NIR region, where water has an extremely strong absorbance. These absorptions indicate the change of dipole moment in bonds and organic compounds that absorb well in the NIR region. Because of the relative weakness of NIR absorption in organic solutions (such as cell culture suspensions), direct measurement is possible without sample preparation or dilution. Overtone absorptions of those fundamental bands occur in the near-infrared (NIR) spectral region, occurring at frequencies some two or three (or more) times the fundamental or first excited vibrational state of the molecular bonds (( 1). The fundamental absorption bands of functional groups occur in the midinfrared region, but the very strong nature of these signals usually requires dilutions to lower absorbances within the linear range of a midinfrared detector. NIRS is an analytical technique based on absorption measured in the near-infrared region (700–2,500 nm) of the electromagnetic spectrum, which is located between the visible and the midinfrared. KEYWORDS: OSMOLALITY, pH, HYDRATED RADII, HYDRONIUM ION, PAT
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WHO SHOULD READ: MANUFACTURING, PROCESS DEVELOPMENT, AND QA/QC
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Near-infrared spectroscopy (NIRS) offers an in situ, real-time monitoring capacity for both of these parameters without the problems associated with on-line pH measurement, providing a closed-loop culture feedback to adjust conditions for improvement of cell performance and productivity. Although in situ pH measurement is possible, it has some inherent technical challenges such as buffer precipitation and glass membrane cell clogging that reduce test result quality. Osmolality is often determined using an off-line osmometer, and pH by potentiometric-based electrodes. To monitor these parameters in cell culture, samples are typically withdrawn and passed through a multifunction analyzer as the BioProfile 400 instrument from NOVA Biomedical ( Withdrawing samples during a bioreactor operation can introduce contamination and lead to sepsis. Osmolality affects cell viability and growth by regulating the transport of water and nutrients through cell membranes and pH maintains the isotonicity of a culture. Osmolality and pH are important cell culture process parameters, key elements that are often examined and optimized to improve the productivity of cell-culture–based bioprocesses.