3_BAS_2024-2025_SamplePreparation-PartII-Preparation(pKa).pdf

Bioanalytical Sciences Sample preparation Importance of pH and pKa Dr. Isabelle Kohler 1 Bioanalytical Sciences | [email protected]  Sample collection, handling and preparation Forensic Toxicology...

Bioanalytical Sciences Sample preparation Importance of pH and pKa Dr. Isabelle Kohler 1 Bioanalytical Sciences | [email protected]  Sample collection, handling and preparation Forensic Toxicology Therapeutic Drug Monitoring Sampling Sample handling & preparation Doping Control Clinical Toxicology & Chemistry Biomarker Discovery Protein precipitation Liquid-liquid extraction Solid-phase extraction 2 Bioanalytical Sciences | [email protected]  Sample collection, handling and preparation Forensic Toxicology Therapeutic Drug Monitoring Sample handling & preparation Doping Control Clinical Toxicology & Chemistry Biomarker Discovery Protein precipitation Liquid-liquid extraction Solid-phase extraction 3 Bioanalytical Sciences | [email protected]  Sample preparation | Importance of the pKa and pH O OH pKa = 2.79 Dissociation constant OH When the pH (of a solution) is equal to Salicylic acid Salicylate the pKa of the analyte, the concentration of the protonated form is equal to the 50% 50% concentration of the deprotonated form At pH 2.79 4 Bioanalytical Sciences | [email protected]  Sample preparation | Importance of the pKa and pH And at other pH values? At pH 0.79 99% AH 1% A- At pH 1.79 90% AH 10% A- At pH 2.79 50% AH 50% A- Rule of thumb: At pH 3.79 10% AH 90% A- A functional group is ≥99% ionized/neutral at At pH 4.79 1% AH 99% A- pH = pKa ± 2 At pH 5.79 0.1% AH 99.9% A- 5 Bioanalytical Sciences | [email protected]  Percentage of ionized form Acid Base 99% of the acid group is ionized 99% of the basic group is ionized when when pH = pKa + 2 pH = pKa - 2 99% of the acid group is neutral 99% of the basic group is neutral when when pH = pKa - 2 pH = pKa + 2 6 Bioanalytical Sciences | [email protected]  Percentage of ionized form Acid Base pH = pKa + 2 pH = pKa - 2 99% ionized 99% ionized 1% ionized 1% ionized pH = pKa - 2 pH = pKa + 2 7 Bioanalytical Sciences | [email protected]  Percentage of ionized form | Chemicalize Create an account (free if limited to molecules with < 12 http://chemicalize.com non-hydrogen atoms) 8 Bioanalytical Sciences | [email protected]  Q&A INTERMEZZO pH and pKa | amphetamine I am interested in extracting amphetamine from urine using SPE, and need the compound to be 99% ionized. To which pH value do I need to adjust my urine sample to have 99% of the compound under its ionized form? If my sample has a pH of 9.3, what is percentage of amphetamine that is under its neutral form? 9 Bioanalytical Sciences 2020-2021 | [email protected]  I am interested in extracting amphetamine from urine using SPE, and need the compound to be 99% ionized. To which pH value do I need to adjust my urine sample to have 99% of the compound under its ionized form? 10  Q&A INTERMEZZO pH and pKa If my sample has a pH of 9.3, what is percentage of amphetamine that is under its neutral form? 11 Bioanalytical Sciences 2020-2021 | [email protected]  If my sample has a pH of 9.3, what is percentage of amphetamine that is under its neutral form? 12 Bioanalytical Sciences 2020-2021 | [email protected]  Q&A INTERMEZZO pH and pKa | MDMA I am interested in extracting MDMA from urine using SPE, and need the compound to be 99% ionized. To which pH value do I need to adjust my urine sample to have 99% of the compound under its ionized form? The pKa of the amine group of MDMA is 9.9. 13 Bioanalytical Sciences 2020-2021 | [email protected]  Q&A INTERMEZZO pH and pKa pH = pKa - 2 I am interested in extracting MDMA from urine using SPE, and need the compound to be 99% ionized. To which pH value do I need to adjust my urine sample to have 99% of the compound under its ionized form? The pKa of the amine group of MDMA is 9.9. At pH lower than 7.9 (9.9 – 2), MDMA is ≥99% ionized. This means that my urine samples has to have a pH lower than 7.9. The pH of urine can be between 3.5 – 8 (usually 4 – 6.5 in non- doped and healthy people). Even though this pH is sufficient to have MDMA ionized, it is better to adjust the pH with a buffer to be sure that all samples have a similar pH (adjust to a value 14 lower than 7.9, the exact value depends on the application).  Sample preparation | Importance of the pKa and pH O OH pKa = 2.79 OH Fentanyl Phenylalanine Salicylic acid Morphine glucuronide MDMA Paclitaxel (Taxol) 15 Bioanalytical Sciences | [email protected]  Sample preparation | Importance of the pKa and pH Hydroxyectoine 16 Bioanalytical Sciences | [email protected]  Sample collection, handling and preparation Forensic Toxicology Therapeutic Drug Monitoring Sampling Sample handling & preparation Doping Control Clinical Toxicology & Chemistry Biomarker Discovery Protein precipitation Liquid-liquid extraction Solid-phase extraction 17 Bioanalytical Sciences | [email protected]