Spectrophotometry Basics

A device within a spectrophotometer that separates light emitted from a lamp into different wavelengths. It uses prisms or gratings to accomplish this.

Gratings, with their fine etched lines, provide more uniform and precise separation of wavelengths compared to prisms.

Slits select a narrow range of wavelengths from the separated light beam, allowing only specific wavelengths to pass through to the sample.

A specialized container that holds the sample to be analyzed in a spectrophotometer. It is usually made of transparent material and has a specific path length.

Polystyrene (325-750nm), PMMA (245-750nm), and quartz (170-2500nm) are typical materials, each with a different range of light transmission.

The typical path length for a cuvette is 1.00cm, allowing light to travel through a specific distance.

They are inexpensive and disposable, suitable for general laboratory use.

Cuvettes that transmit light at shorter wavelengths (e.g., quartz) are more expensive than those that transmit only longer wavelengths (e.g., polystyrene).

A small, transparent container used to hold samples in a spectrophotometer, ensuring proper positioning for light absorption measurements.

A type of spectrophotometer that measures absorbance using very small sample volumes (1-2.5 uL) by compressing the sample between two pedestals and using fiber optic cables for light delivery and detection.

A type of detector commonly used in spectrophotometers to amplify and convert light signals into electrical signals for measurement.

A reference solution containing all components of the sample except the analyte being measured, used to remove background absorbance and ensure accurate measurements.

A spectrophotometer that splits the light beam into two paths, one for the sample and one for the blank, allowing for simultaneous measurement to improve accuracy.

The intensity of light passing through the blank solution in a spectrophotometer, representing the initial light intensity before interacting with the sample.

The intensity of light passing through the sample solution in a spectrophotometer, representing the amount of light reaching the detector after interacting with the sample.

A measure of the amount of light absorbed by a sample at a specific wavelength, expressed as the logarithm of the ratio of incident light intensity (I0) to transmitted light intensity (I).

A fundamental relationship in spectrophotometry that describes how absorbance is related to the concentration of the analyte, the path length of the light beam through the sample, and the molar absorptivity of the analyte.

A graph that depicts the response of a spectrophotometer to known concentrations of an analyte, allowing for the determination of unknown concentrations based on the instrument's response.

The wavelength at which a molecule exhibits the highest absorbance in its absorption spectrum.

A specific group of atoms within a molecule that absorbs light, often containing conjugated double bonds, facilitating electron excitation in the UV-Vis region.

The inherent ability of certain amino acids, like tryptophan, tyrosine, and phenylalanine, to absorb light in the UV-Vis region, allowing for protein quantification.

A prosthetic group found in some proteins, like myoglobin and hemoglobin, that exhibits strong absorption in the UV-Vis region due to its iron-containing structure.

Proteins containing heme groups, involved in electron transport chains, essential for energy production by facilitating redox reactions.

Chemical reactions involving the transfer of electrons between molecules, crucial for energy production and other cellular processes. Think of them as electron exchange.

A series of protein complexes embedded in membranes, where electrons are passed from one molecule to another, releasing energy used to generate ATP, the cell's energy currency.

Non-protein molecules that assist enzymes in specific reactions, often carrying electrons or other groups.

Important coenzymes involved in redox reactions, like NAD and NADH, which are essential for cellular metabolism.

Enzymes that catalyze redox reactions, often involving NAD and NADH, essential for breaking down molecules and producing energy.

A technique used to analyze molecules based on their light absorption properties, helpful for identifying and quantifying biomolecules.

Building blocks of DNA and RNA, containing nitrogenous bases, sugar, and phosphate groups, absorb UV light effectively. These molecules are crucial for genetic information.

Organic molecules containing nitrogen, found in DNA and RNA, primarily responsible for UV light absorption.

A graph showing how much light a molecule absorbs at different wavelengths, reveals its chemical composition and properties.

Liquids in which molecules dissolve, affecting their stability and absorption properties by altering the energy differences between their states.

The addition of a proton (H+) to a molecule, altering its structure and absorption properties by changing its charge and distribution of electrons.

The state of a molecule indicating its level of oxidation or reduction, influencing its absorption properties.

The increase in light absorption of single-stranded DNA compared to double-stranded DNA, a useful tool for studying DNA structure and stability.

A method for measuring protein concentration based on the interaction of a light-absorbing molecule (dye) with proteins in a sample. The dye exhibits a color change upon binding to protein, allowing for quantification.

A protein assay that alters the protein molecule during the measurement process, making the sample unusable for further experiments.

An older colorimetric protein assay based on the reaction of Cu2+ ions with peptide bonds in proteins, resulting in a color change.

A more sensitive colorimetric protein assay that utilizes the initial Cu2+ reaction with peptide bonds, followed by the reduction of Folin-Ciocalteu reagent, producing a distinct color change.

A sensitive and widely used colorimetric protein assay that employs Coomassie Brilliant Blue G-250 dye, which binds to proteins under acidic conditions, causing a color change proportional to protein concentration.

A dye used in the Bradford assay that binds to proteins under acidic conditions. Its color changes depending on its interaction with protein molecules.

The primary type of interaction between Coomassie Blue and protein in the Bradford assay, involving the binding of the dye to the positively charged side chains of basic amino acids (e.g., lysine, arginine).

The secondary type of interaction between Coomassie Blue and protein in the Bradford assay involving the dye's interaction with hydrophobic regions in protein molecules (e.g., aromatic residues).

The range of protein concentrations that can be accurately measured using a specific assay.

A limitation or disadvantage of a protein assay, such as sensitivity to specific protein types or incompatibility with certain reagents.

A detergent that is often used to denature proteins and is incompatible with the Bradford assay due to its interference with the dye binding.

A detergent similar to SDS that is incompatible with the Bradford assay.

The formation of insoluble protein aggregates, which can interfere with the Bradford assay by limiting the dye's access to the protein.

A colorimetric protein quantitation assay that utilizes the complex formation between copper ions and peptide bonds in an alkaline solution. BCA reagent forms a secondary complex with copper ions, resulting in strong light absorption at 562nm.

The BCA assay offers benefits over other protein quantification methods, including high sensitivity, compatibility with most buffers and detergents, and less protein-to-protein variation.

While very useful, the BCA assay has some drawbacks. It requires a longer time compared to the Bradford assay and is more expensive due to specialized reagents.

A measurement of enzyme activity that involves monitoring the chemical reaction catalyzed by the enzyme. This allows for the quantification of the enzyme's ability to convert substrate to product.

Enzyme assays are crucial for understanding enzyme properties, like kinetics and structure, which are vital in various applications spanning medicine, industry, and research.

Enzyme assays rely on the ability to distinguish between substrate and product molecules. Spectrophotometry is frequently used to detect changes in light absorption that occur as the enzyme reaction proceeds.

Special substrates designed to undergo readily detectable changes in their absorption properties when acted upon by an enzyme. This often involves a colorless substrate transforming into a colored product.

A graphical representation of the changes in the relative abundance of a molecule during an enzymatic reaction. It helps visualize the enzyme's activity by observing the formation of product or disappearance of substrate over time.

If a reaction progress curve exhibits a positive slope, it indicates that the product absorbs more light than the substrate. This means the amount of product is increasing over time.

A negative slope on a reaction progress curve means that the product absorbs less light than the substrate. This indicates the substrate is disappearing as the enzyme reaction proceeds.

The y-axis of a reaction progress curve can use various units, such as absorbance, molarity, or moles. This allows for different ways of quantifying the enzyme's activity.

The spectrophotometer plays a crucial role in enzyme assays. It enables us to measure the absorption properties of molecules, allowing us to track the formation of product or disappearance of substrate during an enzymatic reaction.

The study of enzymes is fundamental to biochemistry, as enzymes are essential for almost all biological processes. Understanding their properties is critical for research, medical applications, and industrial processes.

The slope of a reaction's progress curve represents the rate of the reaction. If the slope is increasing, the reaction rate is increasing. If the slope is decreasing, the reaction rate is decreasing.

A linear line indicates that the amount of product formed over time is constant. This means the reaction is proceeding at a consistent rate.

To accurately measure a reaction, it's crucial to ensure that the changes in product formation over time are consistent and measurable. The slope of the line must be clear and represent the rate of the reaction.

Enzyme units provide a standardized way to quantify the amount of enzyme in a sample. This allows comparisons between experiments, ensuring consistent, accurate data.

Enzymes are highly specific, meaning they typically catalyze only a single reaction or a very similar set of reactions. This specificity arises from the enzyme's three-dimensional structure, particularly the active site.

The active site's three-dimensional structure limits the molecules that can fit and interact with it. Only specific substrates with complementary shapes and properties can bind to the active site and be catalyzed.

Both trypsin and chymotrypsin are serine proteases that break down proteins, but they have different side chain preferences, leading them to cleave proteins at different locations depending on the amino acid sequence.

Michaelis-Menten kinetics describes the relationship between the rate of an enzyme-catalyzed reaction and the substrate concentration. It follows a hyperbolic curve, with a maximum reaction rate (Vmax) when the enzyme is saturated with substrate.

The Km is a measure of the substrate concentration at which the reaction rate is half of the maximum rate (Vmax). It represents the affinity of the enzyme for its substrate.

No, Km is a characteristic of the enzyme under specific reaction conditions. It doesn't change even if you increase or decrease the enzyme concentration.

The Lineweaver-Burke plot transforms the Michaelis-Menten curve into a linear graph, making it easier to determine the Km and Vmax values for an enzyme.

Km is determined by the negative reciprocal of the x-intercept of the line, while Vmax is determined by the reciprocal of the y-intercept.

DNA purification removes contaminants from a sample, ensuring that only pure DNA molecules are available for analysis or manipulation through techniques like PCR or restriction enzyme digestion.

Kits utilize a series of steps that selectively degrade and remove non-DNA components, resulting in a pure DNA sample, making the process easier and standardizing the procedure.

DNA purification is essential for removing contaminants like proteins and salts, ensuring a pure and usable DNA sample for research.

Silica columns are used to bind DNA molecules tightly under specific conditions, allowing contaminants to be washed away.

Changing the salt and pH conditions in the column releases bound DNA, allowing it to be collected for further use.

Pure DNA is crucial for accurate analysis and downstream applications, ensuring that results are not affected by contaminants.

DNA purity is assessed by measuring the absorbance of the sample at 260nm (DNA) and 280nm (protein). A ratio of around 1.8-1.9 indicates good purity.

High-quality DNA is not just pure but also structurally intact, without extensive degradation.

Removing stabilizing proteins during purification leaves DNA fragile, making it susceptible to breakage into smaller fragments.

Moderate fragmentation is acceptable as long as the fragments are large enough to contain complete genes and regulatory regions needed for research.

If DNA is broken into very small fragments, it becomes less likely to contain intact gene sequences, impacting the accuracy of experiments.

Knowing the approximate size of DNA fragments is crucial for assessing its quality and suitability for specific applications.

A simple and widely used technique for separating DNA molecules of different lengths based on their migration through a porous gel matrix.

Agarose is a polysaccharide forming a mesh network in solution. This mesh size can be adjusted to separate different sized molecules.

Charged molecules move in an electric field towards the opposite pole. This movement is influenced by the molecule's charge, size, and the gel's properties.

DNA, being negatively charged, migrates towards the positive pole. Larger DNA molecules move slower through the gel than smaller ones.

Higher agarose concentration creates a tighter mesh, slowing down the migration of larger DNA fragments, allowing better separation

A method used to separate proteins based on their molecular weight. Proteins are denatured and coated with a negative charge, causing them to migrate through a gel matrix under the influence of an electric field. Smaller proteins move faster and farther down the gel.

A technique used to separate and purify proteins based on their interactions with a stationary phase, a solid material packed in a column, and a mobile phase, a solution that flows through the column.

The solid material packed in a column, which interacts with proteins in the mobile phase based on properties like charge, size, or affinity.

The solution that flows through the column, typically containing a mixture of proteins, which interact with the stationary phase.

A type of column chromatography that separates proteins based on their charge. Proteins with opposite charges to the stationary phase bind, while those with the same charge flow through.

A powerful technique that separates proteins based on their specific binding to a ligand attached to the stationary phase. This method relies on high affinity interactions between proteins and ligands.

A method that separates proteins based on their size. Smaller proteins enter pores in the stationary phase, delaying their elution, while larger proteins pass through faster.

A type of IEX stationary phase with positively charged groups, which bind negatively charged proteins.

A type of IEX stationary phase with negatively charged groups, which bind positively charged proteins.

A molecule that binds specifically to a protein in affinity chromatography, typically attached to the stationary phase to capture target proteins.

The process of releasing bound proteins from the stationary phase in column chromatography, typically achieved by changing the mobile phase conditions.

Separate samples collected from the bottom of the chromatography column, representing proteins that eluted at different times.

A synthetic polymer commonly used as the basis for resin beads in ion exchange chromatography and other column chromatography techniques.

An agarose-based polysaccharide frequently used as a matrix for stationary phases in column chromatography, particularly affinity chromatography.

Gel electrophoresis separates DNA molecules based on their size, allowing researchers to analyze and identify different DNA fragments.

DNA is negatively charged and migrates towards the positive electrode. Smaller molecules can pass through more pores in the gel, traveling a shorter distance and moving farther down the gel.

DNA ladders contain DNA fragments of known sizes and serve as a reference for determining the size of an unknown DNA sample by comparing their migration distances on the gel.

Protein purification involves cell disruption, selective isolation based on properties, functional assay to check for desired protein, and evaluation of purity.

The first step in protein purification involves breaking open cells to release their contents, including organelles and proteins, into a solution called a lysate. This process is done at low temperatures to prevent protein degradation.

Different proteins are separated based on their unique characteristics (shape, charge, size, and polarity) using various methods like ammonium sulphate precipitation, size exclusion, ion exchange, and affinity chromatography.

Affinity chromatography is a highly selective technique that uses a specific ligand (a molecule that binds to the target protein) to isolate the protein of interest from a mixture of other proteins.

Functional assays are used to determine the amount of desired protein present in each step of the purification process, often by measuring the activity of the protein of interest.

Specific activity refers to the ratio of enzyme activity to the amount of protein in a sample. An increase in specific activity during purification indicates that the desired protein is becoming more concentrated.

Evaluating protein purity is essential to ensure the success of the purification process, determining the effectiveness of each isolation step and verifying that the desired protein is being isolated with minimal contamination.

Protein purification is a fundamental technique in biochemistry essential for studying the structure, function, and interactions of proteins. It allows researchers to isolate and analyze specific proteins, providing crucial insights into cellular processes and how life works.

Protein degradation can significantly affect the quality of the purified protein, leading to incorrect results and hindering the analysis of its properties and functions. This can impact the success of any research involving protein analysis.

Cells can be disrupted using physical methods like grinding, freeze thawing, or sonication, or chemical methods like detergents or high salt concentrations.

Detergents can disrupt cell membranes by dissolving the lipids, allowing the release of intracellular contents, but they must be chosen carefully to avoid denaturing the target protein.

Protein purification is crucial for obtaining pure samples of proteins, allowing researchers to study their structure, function, interactions, and regulation within cells. This is essential for understanding and manipulating biological processes.

The inherent charge, shape, and size of a protein, determined by its amino acid sequence and folding.

The disruption of a protein's native structure by heat and reducing agents like β-mercaptoethanol, unfolding it into a linear chain.

A surfactant that binds to proteins and gives them a uniform negative charge, enabling separation solely based on size.

A porous matrix used in SDS-PAGE, separating proteins based on their ability to migrate through its pores.

A thin gel layer at the top of the separating gel, concentrating proteins into a tight band for sharper separation.

The main gel layer in SDS-PAGE where proteins are separated according to their molecular weight.

A solution containing reducing agents, SDS, glycerol/sucrose, and dye, used to prepare protein samples for loading onto the gel.

Detecting the separated protein bands using staining methods like Coomassie Blue staining or silver staining.

A common method to visualize proteins on SDS-PAGE, staining bands with a blue dye that binds to proteins.

The movement of charged molecules through a gel matrix under the influence of an electric field.

A conductor that provides an electrical connection to a circuit or a medium, facilitating the flow of electricity.

The speed at which a charged molecule moves through a gel matrix during electrophoresis, influenced by factors like charge, size, and shape.

A measure of the resistance a molecule experiences as it moves through a gel matrix, influenced by its size and shape.

A technique used to purify proteins based on their specific binding to a ligand immobilized on a column.

A short sequence of six consecutive histidine residues added to a protein, enabling its purification using nickel-coated chromatography columns.

The consecutive histidine residues in the His-tag bind strongly to nickel ions immobilized on a chromatography column.

A wash buffer helps to remove unbound proteins from the column, leaving only the tagged protein bound to the nickel.

Elution buffer is used to detach the purified protein from the nickel-coated column by competing for the binding site.

A technique that separates molecules based on their size using a porous column filled with beads.

SEC beads are porous and have channels of varying sizes, allowing only molecules smaller than the channel to pass through.

Smaller molecules enter and exit the bead pores, taking longer to elute, while large molecules bypass the beads and elute first.

SEC helps to clean up a partially purified protein by removing small molecules, such as salts.

A technique used to separate proteins based on their molecular weight by electrophoresis in a gel.

SDS denatures proteins and gives them a uniform negative charge, so they migrate towards the positive electrode according to their size.

SDS-PAGE helps to assess the effectiveness of purification by showing if a single band represents the target protein.

High specificity, efficient purification, and minimal sample loss.

Requires knowledge of a specific ligand for the target protein and potential for ligand degradation.

Simple, non-destructive, and can be used to separate a wide range of molecules.

Limited resolution, meaning it's best for separating molecules with significant size differences.

SDS-PAGE stands for Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. It's a technique used to separate proteins based on their size.

Proteins in SDS-PAGE are separated by size. Smaller proteins move faster through the gel, ending up lower. Larger ones move slower, staying higher.

A single band on an SDS-PAGE gel means the sample contains a pure protein, with only one type of protein present.

Multiple bands imply the sample has different types of proteins. Each band represents a different protein size.

Carbohydrates play a range of roles, including energy storage (like starch), structural support (like cellulose), and cell-to-cell communication.

Monosaccharides are simple sugars, the building blocks of carbohydrates. They are single sugar units.

Polysaccharides are complex carbohydrates formed by linking many monosaccharides together. They can be straight or branched chains.

Reducing sugars have a free carbonyl group (aldehyde or ketone) that can react with oxidizing agents. Non-reducing sugars lack this free group.

DNS (3,5-dinitrosalicylic acid) is a reagent that changes color when it is reduced. Reducing sugars can reduce DNS, while polysaccharides cannot.

Triacylglycerols are important for energy storage in plants and animals. They are like fat reserves that can be broken down for energy.

The iodine number is a measure of the degree of unsaturation in a lipid. It tells us how much iodine (a measure of unsaturation) a sample can absorb.

The iodine number helps determine the quality and taste of food. Highly saturated triglycerides are less desirable for health reasons.

The iodine number affects the stability of biofuels. Different plant sources have varying degrees of saturation, impacting fuel quality.

Lipids are a class of organic molecules that are typically not soluble in water. They play essential roles in cell membranes, energy storage, and signaling.

Fatty acids are long chains of carbon atoms with a carboxyl group at one end. They are the building blocks of lipids.

A measure of the degree of unsaturation in a lipid. It represents the amount of iodine, in grams, that is required to saturate 100 grams of the lipid.

Fatty acids containing at least one carbon-carbon double bond. These bonds allow for the addition of iodine.

Fatty acids that contain only single carbon-carbon bonds. They are already 'saturated' and do not react with iodine.

Potassium iodide is added to react with any unreacted iodine chloride (ICl) to form molecular iodine (I2), which can then be titrated with sodium thiosulfate.

A titration method used to determine the amount of iodine in a solution. Sodium thiosulfate reacts with iodine, changing the color of the solution from red/orange to colorless.

A separation technique used to separate components of a mixture based on their relative affinities for a stationary and mobile phase.

A ratio that represents the distance traveled by a solute relative to the distance traveled by the solvent front in paper chromatography.

Molecules that have an uneven distribution of charge, making one end of the molecule more positive and the other end more negative.

Molecules that have an even distribution of charge, making the molecule neutral.

A substance that absorbs certain wavelengths of light and reflects others, giving an object its color.

A green pigment found in plants that absorbs light energy to carry out photosynthesis.

A group of pigments that are responsible for yellow, orange, and red colors in plants.

A series of alternating single and double bonds in a molecule.