Bioanalytical Techniques: Spectrophotometry, Chromatography, Electrophoresis, and Biophysical Methods

Vocabulary practice flashcards covering spectrophotometry, centrifugation, chromatography, electrophoresis, blotting, hydrodynamics, flow cytometry, protein folding thermodynamics, advanced microscopy, and radioactive tracer techniques.

UV–Visible Spectrophotometry

An analytical technique that measures the absorption of ultraviolet ($200\text{--}400\,\text{nm}$) and visible ($400\text{--}700\,\text{nm}$) light as molecules undergo electronic transitions between energy levels.

Beer–Lambert Law

A law stating that absorbance is directly proportional to the concentration of the absorbing substance and the optical pathlength, expressed by the equation $A = \varepsilon b c$.

Molar Extinction Coefficient ($\varepsilon$)

A measurement of how strongly a substance absorbs light at a given wavelength, representing the absorbance produced by a one molar solution under defined conditions and expressed in $\text{L}\,\text{mol}^{-1}\,\text{cm}^{-1}$.

Colorimetry

An optical method that measures the concentration of a substance by assessing the intensity of color produced in solution, primarily using filters and visible light.

Fluorimetry

A sensitive analytical technique that measures fluorescent light emitted by excited molecules as they return to lower energy states following excitation by higher-energy radiation.

Stokes Shift

The difference between the excitation wavelength and the longer emission wavelength in fluorescence.

Infrared Spectroscopy

A technique studying molecular absorption of infrared radiation ($4000\text{--}400\,\text{cm}^{-1}$ in the mid-IR) that produces stretching and bending vibrations accompanied by changes in molecular dipole moments.

Fingerprint Region

The characteristic region of an infrared spectrum that produces unique spectral patterns used to identify specific organic compounds.

Chemical Shift

The resonance position of a nucleus relative to an internal reference standard in NMR spectroscopy, expressed in parts per million ($\text{ppm}$) and determined by surrounding electron shielding.

MALDI-TOF

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight; a soft ionization mass spectrometry method in which laser-excited matrix molecules assist analyte ionization before ions are separated by their flight times under vacuum.

Relative Centrifugal Force (RCF)

The effective centrifugal force exerted on particles relative to Earth's gravitational acceleration, dependent on rotor speed and distance from the rotational axis.

Svedberg Unit ($\text{S}$)

A unit used to quantify particle sedimentation rates in a centrifugal field, where $1\,\text{S} = 10^{-13}\,\text{seconds}$.

Differential Centrifugation

A preparative centrifugation technique that separates cellular organelles and debris based on sedimentation rate through repeated centrifugations at progressively higher speeds.

Isopycnic Centrifugation

A density gradient centrifugation method that separates particles purely based on buoyant density, where particles migrate until reaching a gradient position matching their own density.

Analytical Ultracentrifugation

An ultracentrifugation approach utilizing optical detection systems to monitor the sedimentation behavior of macromolecules in real time to study molecular mass, shape, and interactions.

Stationary Phase

The fixed phase in chromatography (such as paper, silica, alumina, or porous resin) that provides surfaces or chemical groups for differential sample interaction.

Mobile Phase

The liquid, solvent mixture, or gas that moves through or across the stationary phase to transport sample molecules in chromatography.

Retention Time

The time required for a separated compound to travel through a chromatographic column to the detector.

Retardation Factor ($R_f$)

The ratio of the distance travelled by a compound to the distance travelled by the solvent front in planar chromatography, calculated as $R_f = \frac{\text{Distance travelled by compound}}{\text{Distance travelled by solvent front}}$.

Thin-Layer Chromatography (TLC)

A planar chromatography method using an adsorbent stationary phase (such as silica gel or alumina) coated onto a flat plate, separating compounds predominantly through adsorption and partition.

Gel Filtration Chromatography

A size-exclusion separation method where porous beads retard small molecules that enter internal pores while excluding larger molecules, causing the largest molecules to elute first.

Void Volume ($V_0$)

The liquid volume outside the porous beads of a gel filtration column, corresponding to the elution volume of completely excluded, large macromolecules.

Cation-Exchange Chromatography

A form of ion-exchange chromatography featuring negatively charged matrix groups that reversibly bind positively charged target molecules.

Anion-Exchange Chromatography

A form of ion-exchange chromatography featuring positively charged matrix groups that reversibly bind negatively charged target molecules.

Isoelectric Point ($\text{pI}$)

The specific pH at which a protein carries a net electrical charge of zero.

Affinity Chromatography

A highly selective separation technique based on specific, reversible biological interactions between target molecules and immobilized ligands on a solid matrix.

Reverse-Phase HPLC

A high-performance liquid chromatography mode that utilizes a nonpolar stationary phase and a polar mobile phase, causing hydrophobic compounds to be retained longer.

Native PAGE

Polyacrylamide gel electrophoresis conducted in the absence of denaturing agents, separating proteins according to a combination of their native charge, size, and shape.

SDS-PAGE

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis; an electrophoretic method in which proteins are denatured and coated with SDS to yield uniform charge-to-mass ratios, separating them primarily by molecular mass.

Isoelectric Focusing (IEF)

An electrophoretic technique that separates proteins according to their isoelectric points ($\text{pI}$) along an established, stable pH gradient until each protein reaches zero net charge.

Two-Dimensional Electrophoresis

A high-resolution protein separation method combining isoelectric focusing (separation by $\text{pI}$) in the first dimension with SDS-PAGE (separation by molecular mass) in the second dimension.

Pulsed-Field Gel Electrophoresis (PFGE)

An electrophoretic method that resolves very large DNA fragments by periodically alternating the direction of the applied electric field to exploit differences in reorientation times.

Southern Blotting

A molecular biology method used to detect specific DNA sequences by transferring agarose gel-separated DNA to a membrane and probing with a labeled complementary nucleic-acid sequence.

Northern Blotting

A technique used to detect specific RNA sequences and evaluate gene expression by transferring electrophoresed RNA onto a membrane and hybridizing with a labeled complementary probe.

Western Blotting

An analytical method used to detect specific proteins by transferring SDS-PAGE-separated proteins to a membrane and probing with target-specific antibodies.

Viscosity ($\eta$)

The internal resistance offered by a liquid against flow, which generally decreases as temperature increases.

Newtonian Fluid

A fluid whose viscosity remains constant regardless of changes in applied shear conditions or shear rate (e.g., water).

Non-Newtonian Fluid

A fluid whose viscosity changes depending on applied shear rate and shear conditions (e.g., blood).

Intrinsic Viscosity ($[\eta]$)

The macromolecular contribution to solution viscosity extrapolated to zero concentration, related to molecular weight via the Mark-Houwink equation ($[\eta] = K M^a$).

Hydrodynamic Focusing

The fluidics process in flow cytometry where sheath fluid constricts the core sample stream, forcing cells to align in single file through the laser interrogation point.

Forward Scatter (FSC)

The light scattered at narrow angles along the path of the laser beam in flow cytometry, primarily indicating cell size.

Side Scatter (SSC)

The light scattered at approximately $90^\circ$ to the laser beam in flow cytometry, primarily indicating internal cellular granularity and complexity.

Fluorescence-Activated Cell Sorting (FACS)

A specialized flow cytometry application that physically sorts and isolates selected cell populations into containers using electrostatic deflection of droplet-encased cells.

Gibbs Free Energy ($G$)

A thermodynamic property defined by $\Delta G = \Delta H - T\Delta S$, where $\Delta G < 0$ indicates a thermodynamically favorable or spontaneous process under constant temperature and pressure.

Configuration

The permanent spatial geometry of a molecule (such as a protein amino-acid sequence) that can only be altered by breaking and reforming covalent bonds.

Conformation

The flexible spatial arrangement or folded shape of a molecule produced through rotation around single bonds without covalent bond breaking.

Hydrophobic Effect

The thermodynamic tendency of nonpolar groups to cluster together in water to minimize water contact, serving as a primary driving force for protein folding and membrane assembly.

Levinthal's Paradox

The observation that because a polypeptide chain possesses an astronomical number of possible conformations, folding cannot occur by random sampling and must follow biased, directed pathways.

Molecular Chaperones

Specialized proteins (such as Hsp70, Hsp90, and GroEL/GroES) that prevent aggregation and assist nascent or stressed polypeptides in attaining proper folding without becoming permanent components of the folded product.

Phase Contrast Microscopy

An optical microscopy technique that employs phase plates to transform differences in light phase caused by transparent, unstained specimens into visible variations in image brightness.

Dark-Field Microscopy

A microscopy technique that prevents direct light from entering the objective lens, allowing only light scattered by the specimen to form a bright image against a dark background.

Confocal Microscopy

A fluorescence imaging method using a spatial pinhole to reject out-of-focus light from specimens, enabling sharp optical sectioning and three-dimensional reconstruction.

Förster Resonance Energy Transfer (FRET)

A non-radiative transfer of excitation energy from a donor fluorophore to a nearby acceptor fluorophore, used to detect molecular proximity and protein-protein interactions within cells.

Fluorescence Recovery After Photobleaching (FRAP)

An optical technique in which a fluorescent region is bleached with intense light to measure the rate at which surrounding unbleached molecules diffuse back into the bleached area.

Scanning Electron Microscopy (SEM)

An electron microscopy technique that rasters an electron beam across the surface of a specimen to produce high-resolution, three-dimensional-appearing images of surface morphology.

Transmission Electron Microscopy (TEM)

An electron microscopy technique that transmits an electron beam through an ultrathin specimen section to resolve fine internal cellular ultrastructure.

Half-Life ($t_{1/2}$)

The time required for one-half of the unstable nuclei within a radioactive sample to undergo spontaneous radioactive decay.

Phosphorus-32 ($^{32}\text{P}$)

A radioactive isotope emitting energetic beta particles that is widely utilized in molecular biology for labeling DNA and RNA and studying phosphorylation.

Sulfur-35 ($^{35}\text{S}$)

A radioactive beta-emitting isotope incorporated into cysteine and methionine to radiolabel and trace newly synthesized proteins.

Scintillation Counting

A radioactivity measurement technique wherein emitted radiation interacts with a scintillation medium to produce flashes of light that are electronically quantified by photodetectors.

Autoradiography

An imaging method that visualizes the spatial distribution of radiolabeled molecules within gels or tissue sections by exposing them directly to photographic film or phosphor imaging plates.

Geiger-Müller Counter

A gas-filled radiation detection instrument that detects and counts ionizing radiation pulses for routine laboratory radiation-safety monitoring and contamination checks.