Lecture PPT_Bioanalytical CHEM 241 Spring 2024 PDF

Chapter xxx Introduction to Bioanalytical Chemistry Reference Textbooks Lehninger Principles of Biochemistry (PoB) Nelson, David L. (David Lee), author.; Cox, Michael M., author.; Lehninger, Albert L. 2017 Tools and Trends in Bioanalytical Chemistry (TBAC) Kubota, Lauro Tatsuo, editor. 2022 2 Overvi...

Chapter xxx Introduction to Bioanalytical Chemistry Reference Textbooks Lehninger Principles of Biochemistry (PoB) Nelson, David L. (David Lee), author.; Cox, Michael M., author.; Lehninger, Albert L. 2017 Tools and Trends in Bioanalytical Chemistry (TBAC) Kubota, Lauro Tatsuo, editor. 2022 2 Overview PoB 1-2: Biomolecules, Chapter reference: The Foundations of Biochemistry: Chemistry Foundation TBAC 1: Bioanalytical Chemistry TBAC 3: Biosample, Chapter reference: Sampling and Sample Preparation in Bioanalysis Chapter xxx Introduction to Bioanalytical Chemistry Learning Objectives Recognize common functional groups in biomolecules Describe major classes of biomolecules Identify appropriate biosamples for different classes of biomolecules Explain green chemistry principles Describe green chemistry concepts applied to bioanalytical chemistry 3 4 Biomolecules PoB 1.2 The Foundations of Biochemistry: Chemistry Foundation Elements essential to animal life and health. Bulk elements – Structural components of cells and tissues (orange-colored elements) Trace elements – Essential to life, usually because they are essential to the function of specific proteins, including enzymes. – Oxygen-transporting capacity of the hemoglobin molecule requires four iron ions that make-up only 0.3% of its mass (yellow-colored elements) https://themedicalbiochemistrypage.org/hemoglobin-and-myoglobin/ 5 Biomolecules PoB 1.2 The Foundations of Biochemistry: Chemistry Foundation Common Functional Groups – Most biomolecules are derived from hydrocarbons. – Hydrogen atoms are replaced by functional groups, giving rise to a wide array of organic compounds. Typical functional groups – – – – Alcohols (one or more hydroxyl groups) Amines (amino groups) Aldehydes and ketones (carbonyl groups) Carboxylic acids (carboxyl groups) 6 Biomolecules PoB 1.2 The Foundations of Biochemistry: Chemistry Foundation Biomolecules Are Compounds of Carbon with a Variety of Functional Groups Many biomolecules are polyfunctional, containing two or more different kinds of functional groups, each with its chemical characteristics and reactions. The physical-chemical properties of a compound is determined by the chemistry of its functional groups and their disposition in three-dimensional space FIGURE 1–16 Several common functional groups in a single biomolecule. Acetylcoenzyme A (often abbreviated as acetyl-CoA) is a carrier of acetyl groups in some enzymatic reaction 7 Biomolecules Major Classes of Biomolecules Major Classes of Biomolecules – The major classes of biomolecules that are important for all living things are carbohydrates, lipids, proteins, and nucleic acids. – Large biomolecules are called macromolecules 8 Biomolecules PoB 1.2 The Foundations of Biochemistry: Chemistry Foundation Structural hierarchy in the biomolecular organization of cells – In this plant cell, the nucleus is an organelle containing several types of supramolecular complexes, including chromosomes. – Chromosomes consist of macromolecules of DNA and many different proteins. – Each type of macromolecule is made up of simple subunits, i.e. DNA of nucleotides (deoxyribonucleotides) 9 Biomolecules PoB 1.2 The Foundations of Biochemistry: Chemistry Foundation Cells Contain a Universal Set of Small Biomolecules – The aqueous phase (cytosol) of all cells contains a diverse collection of small organic molecules – Consists of 100 to 200 central metabolites crucial for major metabolic pathways in cells. Composition of Biomolecules – Includes common amino acids, nucleotides, sugars, phosphorylated derivatives, and mono-, di-, and tricarboxylic acids. – These biomolecules are polar or charged, watersoluble, and have molecular weights ranging from ~100 to ~500 Da. Concentration of biomolecules – Present in mol to mmol levels within cells. Do not need to remember animal and plant cells 10 Biomolecules PoB 1.2 The Foundations of Biochemistry: Chemistry Foundation Cells Contain a Universal Set of Small Biomolecules Specialized Metabolites – Certain cells or organisms contain specific small biomolecules beyond the universal set. – Example: Vascular plants contain secondary metabolites with roles specific to plant life, such as scents and compounds with physiological effects on humans Summary – The collection of small organic molecules within cells, known as the metabolome, constitutes essential components for cellular metabolism and reflects the evolutionary conservation of metabolic pathways. Do not need to remember animal and plant cells 11 Biomolecules PoB 1.2 The Foundations of Biochemistry: Chemistry Foundation Macromolecules Are the Major Constituents of Cells – Many biomolecules are macromolecules (polymers of high molecular weight assembled from monomers) – Proteins, nucleic acids, and polysaccharides are produced by the polymerization of relatively small compounds with molecular weights of 500 or less Da. – The number of polymerized units can range from tens to millions. – Macromolecules themselves may be further assembled into supramolecular complexes, forming functional units such as ribosomes 12 Biomolecules PoB 1.2 The Foundations of Biochemistry: Chemistry Foundation Macromolecules Are the Major Constituents of Cells Proteins are polypeptides - polymers of amino acids – Proteins are biopolymers made from amino acids linked through peptide bonds in one or more chains of polyamides (polypeptides), which start at the N-terminal and end at the C-terminal. – Proteins show a defined number of monomers per chain, high molecular masses expressed in kDa, and characteristic isoelectric points (pI) 13 Biomolecules PoB 1.2 The Foundations of Biochemistry: Chemistry Foundation Macromolecules Are the Major Constituents of Cells Polysaccharides (large carbohydrates) are polymers of monosaccharides – Carbohydrates are polar molecules that occur as free, such as mono-, oligo-, and polysaccharides, or as covalently bonded to other biomolecules in the form of bioconjugates, with lipids (glycolipids) or proteins (glycoproteins and proteoglycans) 14 Biomolecules PoB 1.2 The Foundations of Biochemistry: Chemistry Foundation Macromolecules Are the Major Constituents of Cells Fats (lipids) are built from fatty acids and glycerol – Lipids are a very diverse group of molecules that are defined as organic molecules soluble in chloroform. – There are many organic compound classes, such as fatty acids and alcohols and their esters, mono-, di-, and triglycerides, sterols, many phospholipids derived from phosphoric acids and esterified with fatty acids – Amphiphilic lipids show dual nature with hydrophilic parts (head group) and lipophilic parts (tails) linked with some polyol 15 Biomolecules PoB 1.2 The Foundations of Biochemistry: Chemistry Foundation Macromolecules Are the Major Constituents of Cells Nucleic acids are polymers of nucleotides – Nucleic acids are fundamental biomolecules for the maintenance of life. – DNA and RNA are polymers, made up of monomers known as nucleotides. When these monomers combine, the resulting chain is called a polynucleotide (poly- = "many"). – Each nucleotide is made up of three parts: a nitrogen-containing ring structure called a nitrogenous base, a five-carbon sugar (a pentose), and at least one phosphate group. 16 Biomolecules A classification of Biomolecules (size) Small molecules: – Includes: lipids and derivatives; vitamins; hormones and neurotransmitters; and carbohydrates. Monomers: – The term monomer refers to compounds which act as building blocks to construct larger molecules called polymers and includes: amino acids; nucleotides; and monosaccharides. Polymers: – Constructed of repeating linked structural units or monomers, polymers (derived from the Greek words polys meaning many and meros meaning parts) include: peptides / oligopeptides / polypetides / proteins; nucleic acids; and oligosaccharides / polysaccharides 17 BioAnalytical Chemistry Targets the qualitative and quantitative analysis of biological activity and detection of biological molecules Uses chemical, biochemical, and instrumental techniques and methods to analyze systems biology. https://link.springer.com/chapter/10.1007/978-3-030-82381-8_1 Sample Preparation ❑ Sample preparation is essential before analysis to… 1) Protect analytical equipment against contamination / wearing 2) Reduce the complexity of samples, by removing interferences from matrix 3) Increase the sensitivity of detection especially for low amounts of analyte ❑ Sample preparation is usually very time consuming… collection, extraction, isolation, pre-concentration, can take up to 80% of total analysis time Example Analyte extraction depends on sample mixture phase 19 Sample Preparation Extraction is the transfer of a solute from a solid or liquid into a different solvent or phase. It enables the isolation/purification or concentration of a desired analyte, or its separation from other species that would interfere with its analysis. Extraction techniques vary with respect to: (i) the nature of the analyte (abundance; physical state, sample size and sampling constraints), (ii) the extraction mechanism Soxhlet Image credit: Daniel Dabbs SPE → Solid-Liquid extraction (e.g. Soxhlet) → Liquid-Liquid Extraction (LLE) → Solid-Phase Extraction (SPE) LibreTexts libraries LLE LibreTexts libraries 20 Sample Preparation Liquid-Liquid Extraction (LLE) LLE is carried out in a separatory funnel to facilitate the analyte recovery. The sample is distributed or partitioned between two immiscible liquids (or phases) where the analyte and matrix have different solubilities. LLE is commonly used for aqueous samples containing both soluble organic and inorganic species. Socratic Q&A Organic solvents with low polarity are used because these are immiscible with water (diethylether, hexane, toluene, dichloromethane, chloroform, carbon tetrachloride). 21 Sample Preparation Liquid-Liquid Extraction (LLE) The immiscible liquids are layered according to their densities and remain in separate phases when they are mixed in any ratio. Diethyl ether, toluene, and hexane are common solvents that are less dense than water. Chlorinated solvents such as chloroform, dichloromethane, and carbon tetrachloride are denser than water. The basis of the extraction process is that the more polar hydrophilic compounds prefer the aqueous (polar) phase and the more non-polar hydrophobic compounds prefer the organic solvent. Lower density liquid Higher density liquid Higher density liquid fraction Image credits: PRHaney, Wikimedia Commons Photo credits: Lisa Nichols LLE Process variables!!! - Polarity of the organic phase - pH of aqueous phase (for ionizable species) - Presence of complexing agents 22 Sample Preparation Liquid-Liquid Extraction (LLE) Partition Process The partition of a solute, S, between two immiscible phases is an equilibrium process that is governed by the distribution law: S (in phase 1) S (in phase 2) The partition coefficient is the equilibrium constant for the reaction at constant temperature T: Extracting phase (numerator) (eq) (eq) Sample phase (denominator) 23  Toluene Phase 2 [S]2 Liquid-Liquid Extraction (LLE) Water Phase 1 Separation by Extraction [S]’1 Suppose solute S in V1 mL of solvent 1 (water) is extracted with V2 mL of solvent 2 (toluene). (eq) That is, the concentration in phase (1) is reduced by a factor q, after the first extraction: [S]1 (eq)’ = q [S]1_0 ’ (eq) (initial) Consider a second extraction with a fresh volume V2 for phase (2), while maintaining the same phase (1). The concentration of solute remaining in phase (1), after the 2nd extraction, is again reduced by a factor q: [S]1’’ = q [S]1’ = q (q [S]1_0) = q2 [S]1_0 After “n” extractions, with fresh volumes V2, we can write: (initial) [S]1 n extractions = qn [S]1_0 (initial) 24 Separation by Extraction Liquid-Liquid Extraction (LLE) EXAMPLE (1): Solute A has a partition coefficient of 3 between toluene and water, with three times as much in the toluene phase. Suppose that 100-mL of a 0.010 M aqueous solution of A are extracted with toluene. What fraction of A remains in the aqueous phase a) after one extraction with 500 mL is performed or b) After 10 extraction performed with 50 mL One extraction Photo credits: Lisa Nichols Sample Preparation 25 Vacuum manifold Solid-Phase Extraction (SPE) or liquid-solid extraction Uses membranes or small disposable cartridges. Solid extracting phase is maintained between two relatively inert frits. The solid packing material is commonly made from powdered silica (SiO2) modified or not by physical coating or chemical bonding with nonpolar, mildly polar, or hydrophilic moieties. http://www.teknokroma.es/en/Productos/filtrationextractionpurification/6/solid-phase-extraction-spe/106/433/vacuummanifold.aspx Extraction of analyte by surface interaction with solid conditioning loading washing isolation e.g. In octadecyl (C18) bonded silica (aka ODS), the non-polar C18 functional groups attract hydrophobic compounds by van der Waals interactions from the aqueous solution. Analyte interferences 26 Separations - Chromatography Column chromatography separation Chromatography is widely used for the separation, identification, and determination of the chemical components in complex mixtures. It resembles solid phase extraction (SPE) in a way that it uses a column and a solid packing to perform the separation. Chromatography was introduced in 1906 whilst SPE was developed much later in year 1971 as an alternative to LLE. In chromatography, the components of a mixture are separated based on differences in the rates at which they are carried by a mobile phase through a (fixed) stationary phase. The rate at which the solute “band” moves through the column is proportional to the time it spends in the mobile phase. Concentration of species B A Eluent volume Separations - Chromatography The stationary phase is fixed in place either in a column or on a planar surface: 27 Gravity Column Chromatography → Column chromatography: stationary phase held in a narrow tube, and mobile phase forced through the tube by gravity or under pressure (for higher performance). → Planar chromatography: stationary phase supported on a flat plate or in the pores of a paper, and mobile phase moves through it by capillary action or gravity. Nichols, L. (2016). Organic Chemistry Laboratory Techniques. Planar (or Thin Layer) Chromatography In column chromatography, the mobile phase may be a gas, liquid, or supercritical fluid. We recognize three chromatography techniques based on the type of mobile phase in use: - Gas Chromatography (GC) High-Performance Liquid Chromatography (HPLC) Supercritical Fluid Chromatography (SFC) Nichols, L. (2016). Organic Chemistry Laboratory Techniques. 28 Analytical Separation HPLC uses high pressure to force the solvent through the packed column in order to achieve high-resolution separations. Chromatographers generally choose GC over LC when there is a choice because GC is normally less expensive and generates much less waste. Some compounds are not sufficiently volatile for GC, hence the need for LC. Commonly 20-40 °C >50 °C up to 300 °C ku.ac.ae 29 Choice of Chromatographic Technique Solute polarity/sample matrix GC Suitable for volatile/semi-volatile compounds HPLC (separation at near ambient temperature) Suitable for poorly volatile compounds. 30 Limitations of Classical Analytical Chemistry TBAC 4. Introduction 31 Biosamples TBAC 3. Sampling and Sample Preparation in Bioanalysis 3.4 Gaseous Samples in Bioanalytics Rare samples, (commonly sampled in environmental and classical analytical chemistry) Gaseous biological samples – For example, air that is exhaled from the lungs 3.5 Liquid Samples in Bioanalytics Liquid, biological samples – For example, sweat, tears, saliva, mucous, urine, blood, plasma, serum, breast milk, and cerebrospinal fluid Liquid samples may include solid samples – For example, dissolution of a solid 3.6 Solid and Semisolid Samples Solids or semisolids – For example, hair, tissues, cells, and viruses 32 Biosample TBAC 3. Sampling and Sample Preparation in Bioanalysis 3.7 Sampling and Sample Preparation of Small Molecules Alseekh, Saleh, et al. "Mass spectrometry-based metabolomics: a guide for annotation, quantification and best reporting practices." Nature methods 18.7 (2021): 747-756. Small molecules can be found in gaseous, liquid, or solid samples. Approaches that are used to characterize as many small molecules or metabolites within a given biological sample is known as metabolomics. The first step in a metabolomics workflow is the rapid stopping, or quenching, of metabolism and extraction of the metabolites to produce a stable extract. This is especially important in highly metabolically active systems such as cells and tissues, but less so in biofluids such as serum, plasma or urine samples 33 Biosample TBAC 3. Sampling and Sample Preparation in Bioanalysis 3.7 Sampling and Sample Preparation of Proteins Proteins are the most abundant macromolecule in cells, approx. ~50% total dry mass The number of different functional proteins in cells and tissues is much higher than other macromolecules The human body is estimated to contain ~100,000 different proteins The number of protein types in nature is yet to be established. Protein can be isolated from a cell, a tissue, an organ, or a whole organism at a given moment Do not need to remember Introduction to Proteins Structure, Function, and Motion, Second Edition By Amit Kessel, Nir Ben-Tal · 2018 34 Biosample TBAC 3. Sampling and Sample Preparation in Bioanalysis 3.7 Sampling and Sample Preparation of Carbohydrates Each year, photosynthesis converts more than 100 billion metric tons of CO2 and H2O into cellulose and other plant products. Certain carbohydrates (sugar and starch) are a dietary staple in most parts of the world. Insoluble carbohydrate polymers serve as structural and protective elements in the cell walls of bacteria and plants and in the connective tissues of animals. Other carbohydrate polymers lubricate skeletal joints and participate in recognition and adhesion between cells. More complex carbohydrate polymers covalently attached to proteins or lipids act as signals that determine the intracellular location or metabolic fate of these hybrid molecules, called glycoconjugates Examples of carbohydrates from food sources 35 Biosample TBAC 3. Sampling and Sample Preparation in Bioanalysis 3.7 Sampling and Sample Preparation of Lipids The lipophilic fraction of any biological sample comprises all fats, such as triglycerides, fatty acids and alcohols, sterols, waxes, and fat-soluble vitamins Many different bio-samples can contain lipids, for example, serum, cells, tissues, and viruses Image Courtesy of Domingues, P., et al. "AACLifeSci Course Companion Manual Advanced Analytical Chemistry for Life Sciences; 2018." 36 Biosample TBAC 3. Sampling and Sample Preparation in Bioanalysis 3.7 Sampling and Sample Preparation of RNA and DNA DNA extraction is well established for whole blood, blood fractions, buccal cells, fresh and frozen tissue, and paraffin tissue blocks RNA is less stable than DNA and is more difficult to extract intact. Special methods and reagents have been developed that allow for the preservation of RNA in blood and tissue. Table 3.2 summarizes source material for nucleic acid extraction and some of the procedural and methodological issues encountered with each sample type. Ref: Chapter 3. Biological Sample collection, processing storage and information management, Jimmie B. Vaught and Marianne K. Henderson