Lab Techniques, Spectroscopy, and Spectrometry

Question 1

Separations

Answer 1

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

Question 2

What are the types of laboratory separations you should know?

Answer 2

Extraction, distillation, crystallization, and chromatography

Question 3

Extraction

Answer 3

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

Question 4

Common extraction technique in biochemistry

Answer 4

Phenol-Chloroform extraction

Used to separate nucleic acids from cellular proteins

Question 5

Distillation

Answer 5

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

Question 6

Spectroscopy

Answer 6

Study of interaction between matter and electromagnetic radiation (light)
 Includes:
 1. Nuclear Magnetic Resonance (NMR)
 2. Infrared (IR)
 3. Ultraviolet-Visible (UV-Vis)

Question 7

Nuclear Magnetic Resonance (NMR)

Answer 7

Study of interaction between atomic nuclei and radio waves

Question 8

Nuclear Spin

Answer 8

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

Question 9

Low and high energy states of nuclear spin when subject to external magnetic field

Answer 9

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

Question 10

What is varied in NMR?

Answer 10

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

Question 11

What does the x axis on an NMR spectrum represent?

Answer 11

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)

Question 12

What are chemically equivalent hydrogens?

Answer 12

Hydrogens whose positions on a compound are indistinguishable by NMR

Question 13

Chemical Shift

Answer 13

Difference between resonance frequency of chemically shifted hydrogens and resonance frequency of hydrogens on a reference compound such as tetramtehylsilane

Question 14

What is the integral trace in NMR?

Answer 14

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.

Question 15

What is Splitting caused by in NMR?

Answer 15

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

Question 16

What is the chemical shift for aldehyde protons in NMR?

Answer 16

9.5 ppm

Question 17

What is the chemical shift for carboxylic acid protons in NMR?

Answer 17

10-12 ppm

Question 18

What is the chemical shift for benzene protons in NMR?

Answer 18

8 ppm

Question 19

What is the chemical shift for alcohol protons in NMR?

Answer 19

1-5 ppm

Question 20

What is the chemical shift for methyl protons in NMR?

Answer 20

Less than 1 ppm

Question 21

What types of atoms register on an NMR?

Answer 21

Atoms with a nuclear spin, which means odd atomic or mass number

Carbon-13
Hydrogen-1

Question 22

IR Spectroscopy

Answer 22

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

Question 23

Infrared region of electromagnetic spectrum

Answer 23

Frequency just below the visible spectrum of light, with higher wavelengths

Question 24

What happens when compounds with dipoles are exposed to infrared radiation?

Answer 24

Polar bonds within the compound stretch and contract, causing intramolecular vibrations and rotations

Question 25

IR Absorption Graphs

Answer 25

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

Question 26

What are some common peaks to know in IR spectroscopy?

Answer 26

``` 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 ```

Question 27

Ultraviolet region of EM spectrum

Answer 27

Short wavelengths, higher frequency than visible and infrared 200-400 nm wavelengths

Question 28

Ultraviolet Spectroscopy

Answer 28

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

Question 29

UV Spectrum

Answer 29

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

Question 30

How do electrons in conjugated systems absorb light and how is this measured in UV spectroscopy?

Answer 30

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

Question 31

What is the equation for molar absorptivity?

Answer 31

Molar absorptivity = A/cl A: Absorbance c: concentration of sample l: length of path of light through cell

Question 32

When does a compounds absorbance move into the visible region of the EM spectrum?

Answer 32

If the compound has 8 or more double bonds | E.g. beta-carotene, precursor of vitamin A Think carrots

Question 33

Mass Spectrometry

Answer 33

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)

Question 34

Molecular Ion

Answer 34

Largest possible ion created in mass spectrometry | Size of original molecule subtracting just one electron

Question 35

Parent Peak in Mass Spectrometry

Answer 35

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

Question 36

Abundance

Answer 36

Measurement of quantity of a particular ion in a mass spectrometry computed from count of particular ion over base peak count

Question 37

Base Peak in Mass Spectrometry

Answer 37

Largest peak on the mass spectrometry graph | Plots mass to charge ratio (m/Z) against Abundance (counts that hit detector)

Question 38

Spectrometry

Answer 38

Spectrometry: study of interactions between matter and energy sources other than EM radiation

Question 39

Fingerprint region

Answer 39

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

Question 40

When does distillation fail?

Answer 40

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

Question 41

Fractional Distillation

Answer 41

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

Question 42

Crystallization

Answer 42

Technique that purifies solid compounds using the principle that pure substances form crystals more easily than impure substances Can be very inefficient

Question 43

Chromatography

Answer 43

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

Question 44

Column chromatography

Answer 44

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

Question 45

High Pressure Liquid Chromatography (HPLC)

Answer 45

Variant of column chromatography in which column and solution use an apparatus that puts the system under high pressure

Question 46

Paper Chromatography

Answer 46

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

Question 47

Thin-layer chromatography

Answer 47

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

Question 48

Gas-liquid chromatography

Answer 48

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

Question 49

Size-Exclusion Chromatography

Answer 49

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)

Question 50

Ion-exchange chromatography

Answer 50

Molecules are separated based on net surface charge | Utilizes cationic or anionic “exchangers” to slow down movement of charged molecules

Question 51

Affinity Chromatography

Answer 51

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

Question 52

Gel Electrophoresis

Answer 52

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

Question 53

How can proteins be separated using gel electrophoresis?

Answer 53

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)

Question 54

Isoelectric Focusing

Answer 54

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

Question 55

How is a ladder used in gel electrophoresis and for what reason?

Answer 55

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

Question 56

Blotting

Answer 56

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

Question 57

Southern Blotting

Answer 57

Identifies target fragments of a known DNA sequence in a large population of DNA 1. Cleave DNA 2. Gel electrophoresis to resolve pieces according to size 3. Southern blot it onto membrane after denaturing to ssDNA 4. Add complementary D/RNA probe to visualize

Question 58

Northern Blot

Answer 58

Same as Southern Blot, but instead for RNA fragments

Question 59

Western Blot

Answer 59

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

Question 60

How can you resolve enantiomers from racemic mixture?

Answer 60

1. Differences in crystallization of enantiomers- direct visualization of crystals 2. Stereospecific enzymes that can be added and will react only with one enantiomer creating separable compound