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MCAT Biochemistry > Lab Techniques, Spectroscopy, and Spectrometry > Flashcards

Lab Techniques, Spectroscopy, and Spectrometry Flashcards

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MCAT flashcards
1
Q

Separations

A

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

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2
Q

What are the types of laboratory separations you should know?

A

Extraction, distillation, crystallization, and chromatography

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3
Q

Extraction

A

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

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4
Q

Common extraction technique in biochemistry

A

Phenol-Chloroform extraction

Used to separate nucleic acids from cellular proteins

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5
Q

Distillation

A

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

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6
Q

Spectroscopy

A 
Study of interaction between matter and electromagnetic radiation (light)
 Includes:
 1. Nuclear Magnetic Resonance (NMR)
 2. Infrared (IR)
 3. Ultraviolet-Visible (UV-Vis)
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7
Q

Nuclear Magnetic Resonance (NMR)

A

Study of interaction between atomic nuclei and radio waves

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8
Q

Nuclear Spin

A

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

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9
Q

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

A

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

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10
Q

What is varied in NMR?

A

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

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11
Q

What does the x axis on an NMR spectrum represent?

A

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)

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12
Q

What are chemically equivalent hydrogens?

A

Hydrogens whose positions on a compound are indistinguishable by NMR

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13
Q

Chemical Shift

A

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

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14
Q

What is the integral trace in NMR?

A

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.

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15
Q

What is Splitting caused by in NMR?

A

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

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16
Q

What is the chemical shift for aldehyde protons in NMR?

A

9.5 ppm

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17
Q

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

A

10-12 ppm

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18
Q

What is the chemical shift for benzene protons in NMR?

A

8 ppm

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19
Q

What is the chemical shift for alcohol protons in NMR?

A

1-5 ppm

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20
Q

What is the chemical shift for methyl protons in NMR?

A

Less than 1 ppm

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21
Q

What types of atoms register on an NMR?

A

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

Carbon-13
Hydrogen-1

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22
Q

IR Spectroscopy

A

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

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23
Q

Infrared region of electromagnetic spectrum

A

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

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24
Q

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

A

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

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25
Q

IR Absorption Graphs

A

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

26
Q

What are some common peaks to know in IR spectroscopy?

A 
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
27
Q

Ultraviolet region of EM spectrum

A

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

28
Q

Ultraviolet Spectroscopy

A

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

29
Q

UV Spectrum

A

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

30
Q

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

A

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

31
Q

What is the equation for molar absorptivity?

A

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

32
Q

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

A

If the compound has 8 or more double bonds

E.g. beta-carotene, precursor of vitamin A
Think carrots

33
Q

Mass Spectrometry

A

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)

34
Q

Molecular Ion

A

Largest possible ion created in mass spectrometry

Size of original molecule subtracting just one electron

35
Q

Parent Peak in Mass Spectrometry

A

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

36
Q

Abundance

A

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

37
Q

Base Peak in Mass Spectrometry

A

Largest peak on the mass spectrometry graph

Plots mass to charge ratio (m/Z) against Abundance (counts that hit detector)

38
Q

Spectrometry

A

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

39
Q

Fingerprint region

A

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

40
Q

When does distillation fail?

A

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

41
Q

Fractional Distillation

A

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

42
Q

Crystallization

A

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

Can be very inefficient

43
Q

Chromatography

A

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

44
Q

Column chromatography

A

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

45
Q

High Pressure Liquid Chromatography (HPLC)

A

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

46
Q

Paper Chromatography

A

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

47
Q

Thin-layer chromatography

A

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

48
Q

Gas-liquid chromatography

A

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

49
Q

Size-Exclusion Chromatography

A

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)

50
Q

Ion-exchange chromatography

A

Molecules are separated based on net surface charge

Utilizes cationic or anionic “exchangers” to slow down movement of charged molecules

51
Q

Affinity Chromatography

A

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

52
Q

Gel Electrophoresis

A

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

53
Q

How can proteins be separated using gel electrophoresis?

A

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)

54
Q

Isoelectric Focusing

A

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

55
Q

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

A

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

56
Q

Blotting

A

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

57
Q

Southern Blotting

A

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
58
Q

Northern Blot

A

Same as Southern Blot, but instead for RNA fragments

59
Q

Western Blot

A

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

60
Q

How can you resolve enantiomers from racemic mixture?

A
  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

MCAT Biochemistry (11 decks)

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