Lab Techniques, Spectroscopy, and Spectrometry Flashcards
Separations
Variety of lab techniques that use intermolecular forces to separate a mixture into its component parts
Compounds can be separated using: solubility, melting point, boiling point, or any other physical property
What are the types of laboratory separations you should know?
Extraction, distillation, crystallization, and chromatography
Extraction
Separation technique based on solubility
Usually involves two immiscible phases (aqueous and less dense organic layer) due to polarity
Add weak acid to remove strong base, strong acid to remove weak and strong bases, weak base to remove strong acids, and strong base to remove weak and strong acids, always shake and then let aqueous layer drain
Common extraction technique in biochemistry
Phenol-Chloroform extraction
Used to separate nucleic acids from cellular proteins
Distillation
Technique used to separate compounds that have significantly different boiling points
Solution of two volatile liquids with boiling point differences > 20 degree C can be separated by slow boiling
Compound w/ low bp captured and condensed in a cool tube first
Not completely efficient- azeotrope forms
Spectroscopy
Study of interaction between matter and electromagnetic radiation (light) Includes: 1. Nuclear Magnetic Resonance (NMR) 2. Infrared (IR) 3. Ultraviolet-Visible (UV-Vis)
Nuclear Magnetic Resonance (NMR)
Study of interaction between atomic nuclei and radio waves
Nuclear Spin
Nuclei with odd atomic or mass numbers have this property
Spinning nuclei generates magnetic field
When subject to external magnetic field, either aligns with (alpha spin state) or against (beta spin state) external magnetic field
Low and high energy states of nuclear spin when subject to external magnetic field
High energy state occurs when nuclei aligns against magnetic field (beta)
Lower energy state occurs when nuclei aligns with magnetic field
Difference between low and high energy states (delta E) is proportional to magnetic field strength
What is varied in NMR?
The frequency of the EM radiation is held constant, while the magnetic field strength is varied
Magnetic field will need to be larger for a shielded proton
What does the x axis on an NMR spectrum represent?
The magnetic field strength measured in parts per million (ppm)
Increases left to right despite numbers on axis
Leftward is downfield (less shielded due to electron withdrawing groups)
Rightward is upfield (more shielded due to electron donating groups)
What are chemically equivalent hydrogens?
Hydrogens whose positions on a compound are indistinguishable by NMR
Chemical Shift
Difference between resonance frequency of chemically shifted hydrogens and resonance frequency of hydrogens on a reference compound such as tetramtehylsilane
What is the integral trace in NMR?
Line drawn above peaks that rises each time it goes over a peak. Rise is proportional to number of chemically equivalent hydrogens in peak beneath it.
What is Splitting caused by in NMR?
AKA spin-spin splitting
Caused by neighboring hydrogens that are not chemically equivalent
Neighboring: H bound to an atom adjacent to atom to which H of interest is bound
Number of peaks is n+1, n is number of neighboring H’s
What is the chemical shift for aldehyde protons in NMR?
9.5 ppm
What is the chemical shift for carboxylic acid protons in NMR?
10-12 ppm
What is the chemical shift for benzene protons in NMR?
8 ppm
What is the chemical shift for alcohol protons in NMR?
1-5 ppm
What is the chemical shift for methyl protons in NMR?
Less than 1 ppm
What types of atoms register on an NMR?
Atoms with a nuclear spin, which means odd atomic or mass number
Carbon-13
Hydrogen-1
IR Spectroscopy
Uses molecular dipoles to find information about functional groups
Infrared spectrometer slowly changes frequency of infrared light on compound and records frequencies of absorption in number of cycles/cm
Without dipoles, no energy is absorbed
Infrared region of electromagnetic spectrum
Frequency just below the visible spectrum of light, with higher wavelengths
What happens when compounds with dipoles are exposed to infrared radiation?
Polar bonds within the compound stretch and contract, causing intramolecular vibrations and rotations
IR Absorption Graphs
Have lower num cycles/cm to the right and higher cycles/cm to the left
Peaks will appear upside-down
Stiffer and stronger bonds will have peaks farther to the right of the graph
Sp > sp^2 > sp^3
What are some common peaks to know in IR spectroscopy?
C=O sharp dip around 1700 /cm OH broad dip around 3200-3600 /cm Triple bonded C’s sharp dip around 2200 /cm C-O sharp dip around 1100 /cm N-H broad dip (3000-3400?) /cm C-H may be around 2800-3000 /cm
Ultraviolet region of EM spectrum
Short wavelengths, higher frequency than visible and infrared
200-400 nm wavelengths
Ultraviolet Spectroscopy
Detects conjugated systems (double bonds separated by one single bond) by comparing intensities of two beams of light from same monochromatic light source
One shone through sample and other through reference cell
Difference in energy absorbed is recorded as a UV spectrum
UV Spectrum
Shows peaks in regions where conjugated systems of electrons absorbed energy
Usually a 30-40 nm increase for each additional conjugated double bond, 5 nm increase for each additional alkyl group attached to an atom in conjugated system
Carbonyls absorb light in UV region
Plots absorbance to wavelength
How do electrons in conjugated systems absorb light and how is this measured in UV spectroscopy?
Electrons in conjugated systems have close LUMOs to their HOMOs (lowest unoccupied molecular orbital and highest unoccupied molecular orbital), which allow them to be moved up to pi* orbitals easily.
This means measured intensity will be lower in UV spectroscopy as compared to control sample
What is the equation for molar absorptivity?
Molar absorptivity = A/cl
A: Absorbance
c: concentration of sample
l: length of path of light through cell
When does a compounds absorbance move into the visible region of the EM spectrum?
If the compound has 8 or more double bonds
E.g. beta-carotene, precursor of vitamin A
Think carrots
Mass Spectrometry
Determines a compound’s molecular weight, and molecular formula
Molecules of sample bombarded with e- causing ionization
Ions then accelerated through magnetic field- force deflects ions around a curved path
Radius of curvature depends on mass to charge ratio (m/Z)
Molecular Ion
Largest possible ion created in mass spectrometry
Size of original molecule subtracting just one electron
Parent Peak in Mass Spectrometry
Peak made by molecular ion after a computer counts the number of ions that hit the detector
All the way to the right on the spectrum
Can measure abundance of parent peak to base peak
Abundance
Measurement of quantity of a particular ion in a mass spectrometry computed from count of particular ion over base peak count
Base Peak in Mass Spectrometry
Largest peak on the mass spectrometry graph
Plots mass to charge ratio (m/Z) against Abundance (counts that hit detector)
Spectrometry
Spectrometry: study of interactions between matter and energy sources other than EM radiation
Fingerprint region
Region in IR spectrum between 600-1400 /cm where complex vibrations that make a compound unique show up
Can distinguish one compound from a similar compound
When does distillation fail?
If solution of two volatile liquids exhibits positive deviation to Raoult’s law, solution will boil at a lower temperature than either pure compound
Resulting solution will be exact ratio of two liquids
Fractional Distillation
More precise distillation that separates liquids with closer boiling points
Vapor is run through glass beads, allowing compound with higher boiling point to repeatedly condense and fall back into solution
Crystallization
Technique that purifies solid compounds using the principle that pure substances form crystals more easily than impure substances
Can be very inefficient
Chromatography
Can be used to purify a compound from a mixture and/or determine the ratio of compounds in a mixture
Involves separation of a mixture by passing it over or through a matrix that adsorbs (binds) different compounds more or less strongly according to properties
Mobile and stationary phases
Column chromatography
Solution containing mixture is dripped down a column containing solid phase (glass beads made of polar molecule, SiO2)
More polar molecules travel more slowly, creating separate layers
Compounds collected as they elute with solvent and drip out of column
High Pressure Liquid Chromatography (HPLC)
Variant of column chromatography in which column and solution use an apparatus that puts the system under high pressure
Paper Chromatography
Technique in which small portion of sample to be separated is spotted onto paper. One end of paper placed into non-polar solvent, which moves up the paper, bringing along the sample spots. More polar sample compounds will move more slowly
Compute Rf factor by dividing distance traveled by total distance traveled by solvent
Thin-layer chromatography
Similar technique to paper chromatography, except a coated glass or plastic plate is used instead of paper
Results are visualized in iodine vapor chamber
Rf factor computed for sample
Gas-liquid chromatography
Chromatography technique in which stationary phase is liquid and mixture is dissolved into a heated carrier gas and passed over liquid phase bound to a column
Compounds in mixture equilibrate with liquid phase at different rates and exit through port
-graph with peaks of elution times
Size-Exclusion Chromatography
Chromatography well suited for separation of peptides and proteins
Separate by size and sometimes molecular weight through gel filtration with large molecules eluting first (don’t penetrate well in stationary phase, so travel quickly)
Ion-exchange chromatography
Molecules are separated based on net surface charge
Utilizes cationic or anionic “exchangers” to slow down movement of charged molecules
Affinity Chromatography
Technique that uses highly specific interactions to slow down select molecules, rather than separating out molecules with a specific property
E.g. receptor-ligand, enzyme-substrate, antigen-antibody interactions
Gel Electrophoresis
Technique used to separate very specific molecules. Molecular mixture placed in agarose gel and electric field
Nucleic acids will migrate through gel in response to electric field, larger particles move more slowly
Bands can be visualized and examined
How can proteins be separated using gel electrophoresis?
Proteins are usually too large to migrate through pores, so different gel used. Proteins coated and denatured with detergent which coats with negative charge proportional to length
Form bands for visualization
Can also be separated by isoelectric points (pI)
Isoelectric Focusing
Similar to gel electrophoresis- Gel with a stable pH gradient is used and an electric field is applied. Proteins in a pH region below isoelectric point will be positively charged and move towards negatively charged electrode. Will eventually reach point of neutrality in pH gradient and stop moving in gel. Sharp bands shown
How is a ladder used in gel electrophoresis and for what reason?
Ladder is a mixture of DNA, RNA, or polypeptide fragments of known sizes or quantities. Migration distances of samples can be compared to ladder’s bands to determine approximate size of sample components
Blotting
Technique by which molecules are transferred from the gel onto a membrane, maintaining the same spatial relationship of bands based on size
Allows for easier manipulation or visualization of the molecules
Southern Blotting
Identifies target fragments of a known DNA sequence in a large population of DNA
- Cleave DNA
- Gel electrophoresis to resolve pieces according to size
- Southern blot it onto membrane after denaturing to ssDNA
- Add complementary D/RNA probe to visualize
Northern Blot
Same as Southern Blot, but instead for RNA fragments
Western Blot
Used to detect particular proteins in a mixture of proteins
Basic process of transferring resolved protein mixture from gel to a separate membrane is same as Southern or Northern Blotting
Visualize with antibodies: primary antibody bind to protein, secondary binds to primary and has enzyme to catalyze fluorescent product
How can you resolve enantiomers from racemic mixture?
- Differences in crystallization of enantiomers- direct visualization of crystals
- Stereospecific enzymes that can be added and will react only with one enantiomer creating separable compound
