FREE BACE Preparing solutions Flashcards

A biotechnician needs to make 1 L of 50 mM potassium phosphate buffer at pH 6.0 (pKa2 of phosphate = 7.2). Which phosphate salts should be used and what ratio of KH2PO4 to K2HPO4 is needed?

Answer: KH2PO4 and K2HPO4 with approximately 16 parts KH2PO4 to 1 part K2HPO4

Using Henderson-Hasselbalch: 6.0 = 7.2 + log([K2HPO4]/[KH2PO4]), giving [K2HPO4]/[KH2PO4] = 10^(-1.2) = 0.063. So KH2PO4:K2HPO4 ratio is approximately 16:1.

What is the difference between analytical grade and technical grade reagents and when does the distinction matter?

Answer: Analytical grade has high purity (>99%) with known low impurity levels; technical grade has lower purity with less specification. The distinction matters for sensitive assays biological applications and when impurities could interfere with results

Analytical grade (ACS grade) has purity >99% with specified maximum impurity levels; technical/practical grade has lower purity. The distinction matters critically in sensitive biochemical assays, cell culture, and trace analysis.

A standard operating procedure (SOP) requires preparing a solution of NaOH, 1 N for use in a titration. What does normality (N) mean for NaOH?

Answer: Normality equals molarity times equivalents per mole; for NaOH (1 OH- per formula unit) 1 N = 1 M. For H2SO4 (2 H+ per formula unit) 1 N = 0.5 M

Normality = molarity x equivalents per mole. For NaOH (provides 1 OH- per molecule), 1 N = 1 M. For sulfuric acid (provides 2 H+ per molecule), 1 N = 0.5 M.

When preparing a tissue culture media supplement with multiple components, what is the recommended approach for most reliable preparation?

Answer: Prepare concentrated stocks of each component separately validate each independently then combine in proportional volumes to achieve the desired final concentration

Preparing and validating individual concentrated stocks before combining ensures accuracy, allows independent quality control, simplifies troubleshooting, and enables accurate proportional dilution.

What is the purpose of filtering a freshly prepared buffer solution through a 0.22 µm membrane before use in HPLC or cell culture?

Answer: To sterilize the buffer and remove particulates that could clog HPLC columns or introduce microbial contamination into cell cultures

0.22 µm filtration sterilizes the buffer (removes bacteria and fungi) and removes insoluble particles that could cause column back-pressure in HPLC or contaminate cell cultures.

What are endotoxins and why do solution preparations for mammalian cell culture require specific testing for them?

Answer: Endotoxins are lipopolysaccharide (LPS) components of gram-negative bacterial cell walls that contaminate solutions and cause severe inflammatory responses in mammalian cells and animal models

Endotoxins (LPS from gram-negative bacteria) are heat-stable, 0.22 µm-filter-resistant contaminants that cause TLR4-mediated inflammatory signaling in mammalian cells, affecting cell behavior and experimental results.