EK 4: Lab Techniques Flashcards Preview

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Flashcards in EK 4: Lab Techniques Deck (49):
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Extraction (separation technique)

Separation based on?

Apparatus?

How does it work?

What is used?

Steps?

Order of steps and why?

Extraction = separation technique involving solubility differences and an apparatus containing two immiscible (not mixable) fluids.

  • 2 immiscible fluids:
    • Top: Less dense organic (nonpolar) mixture
    • Bottom: Denser aqueous (polar) mixture
  • Separation technique based on solubility:     "Like dissolves like"
    • Nonpolar compounds are soluble in nonpolar mixtures
    • Polar compounds are soluble in polar mixtures
  1. The organic nonpolar top mixture contains multiple nonpolar compounds.
  2. The desired compound is separated from the rest by making it polar.
  3. An acid/base is added to protonate/deprotonate the desired compound, making it polar.
  4. This causes it to dissolve in the aqueous (polar) fluid on bottom.
  5. Then, the bottom aqueous fluid with the desired compound can be drained out of the apparatus.`
  • It is important to use either of the following two orders: (weak → strong →  weak →  strong)
    • Weak acid → Strong acid → Weak base → Strong base
    • Weak base →  Strong base → Weak acid → Strong acid
  • This is because strong bases/acids will make all their compliments (weak or strong) polar, which would cause both weak and strong compliments to be drained out unseparated (not something we want to do).
    • Weak base deprotonates strong acid
    • Strong base deprotonates both strong/weak acid
    • Weak acid protonates strong base
    • Strong acid protonates both strong/weak base

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Distillation (separation technique)

Separation based on?

How does it work? Steps?

Distillation - technique used to separate compounds with different boiling points.

  • Based on differing boiling points
  1. A mixture of the compounds is heated slowly.
  2. The compound with the lower boiling point boils out of the mixture first forming a vapor.
  3. This vapor travels through a cooled tube, which causes the vapor to decrease in temperature and condense back to liquid
  4. Our desired compound is now in liquid. form and captured in a separate container.

 

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Chromatography

Separation based on?

 

Column Chromatography

Paper/Thin-Layer Chromatography

Gas-Liquid Chromatography

Stationary and Mobile phase for each?

Describe each?

Location of most polar and most nonpolar compounds at the end of each?

 

Chromatography - lab technique to separate compounds based on different polarities.

A mixture is passed through a matrix that binds to some compounds stronger than others.

  • The differences in binding strength affect the rate at which the compounds pass through the matrix.
  • Stationary phase: Polar
  • Mobile phase: Nonpolar
  • Thus, polar compounds bind to the stationary phase and move slower.
  • Nonpolar compounds elute out of the matrix faster.

Column Chromatography

  • Stationary phase: glass beads (polar)
  • Mobile phase: liquid (nonpolar)
  • The mobile phase passes through a glass column with glass beads in it.
  • Most polar solvent is on the top because it moves down the slowest.
  • Nonpolar solvent is on the bottom because it moves down the fastest.

Paper/Thin-layer Chromatography

  • Stationary phase: paper (polar)
  • Mobile phase: liquid (nonpolar)
  • A polar paper is placed in a dish of nonpolar liquid containing the compounds.
  • The compounds travel up the paper towards the top.
  • Most polar compound moves up the slowest, thus its at the bottom of the paper
  • Nonpolar compound moves up the fastest, thus its at the top of the paper

Gas-Liquid Chromatography

  • Stationary phase: liquid (polar)
  • Mobile phase: carrier gas (nonpolar)
  • A mixture of compounds is dissolved into a gas, which carries it through a stationary liquid.
  • Most polar compounds move the slowest through the liquid, thus aren't very far through it.
  • Nonpolar compounds move the fastest through the liquid, thus move far through it.

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Size-exclusion chromatography

Ion-exchange chromatography

Affinity chromatography

 

what property does each one use to separate compounds?

Size-exclusion chromatography - compounds are separated based on their sizes and/or molecular weights

Ion-exchange chromatography - compounds are separated based on their charges

Affinity chromatography - compounds are separated based on their affinity to certain things

  • ex: receptors, enzymes, and antibodies that bind to that specific compound are used.

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Gel electrophoresis

Gel Electrophoresis - technique used to separate proteins or nucleic acids (DNA/RNA) based on their size and charge.

 

  1. Proteins or nucleic acids are placed in wells at the top of a gel.
    1. They are both negatively charged, so they migrate towards the anode.
  2. An electrical charge is applied to the gel.
  3. They migrate different distances through the agarose gel due to differing sizes and charges.
  4. Bands are formed and compared to a ladder of known sizes

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Southern Blotting

Southern Blotting - technique used to identify a specific fragment of DNA by using its fluorescent-tagged complementary fragment.

 

  1. A southern blot begins with DNA that has already been cleaved and separated by gel electrophoresis.
  2. The separated bands of DNA are moved onto another membrane.
  3. The fluorescent-tagged complementary fragment is added to the membrane.
  4. It anneals to the DNA fragment of interest and is now visible.

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  • Proteins are too large to pass through the pores in gel electrophoresis.
  • Thus, gel electrophoresis for protein separation is done without the pores.

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3 ways to separate enantiomers in a racemic mixture?

Separating Enantiomers in a Racemic Mixture:

  1. Separation by Crystallization:
    • Each enantiomer forms a different crystal
  2. Separation using Stereospecific Enzymes:
    • There are enzymes that modify only one of the enantiomers.
    • Then they can be separated.
  3. Separation by Conversion to Diastereomers:
    • Reactions are done to convert the enantiomers to diastereomers, which can be separated.
      • Enantiomers have same physical/chemical properties.
      • But Diastereomers have different physical/chemical properties.

 

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DNA is negative due to negatively charged phosphate groups.

 

Anions (-) move toward the Anode (+)

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Nuclear Magnetic Resonance (NMR) Spectroscopy

  1. How does it work? Only works for compounds with odd or even # of protons?
  2. Chemical shift? Units?
  3. How do you determine the # of peaks for a certain proton?
  4. How do you determine the area under the peak?
  5. What happens if the proton is near an electron withdrawing group? shielding? deshielding?

**For the following spectra, label which peak belongs to A, B, C?

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NMR Spectroscopy:

"Nuclear" refers to Protons

"Magnetic" refers to an External Magnetic field

"Resonance" refers to Absorbance of radio waves

 

Deals with the magnetic spin of hydrogen nuclei.

  • Only works for compounds with odd # of protons, because a proton must be alone so that an external magnetic field can affect its spin.
    • even # protons are paired together and already spin opposite of each other.
  • NMR spectroscopy - technique involving radio waves and protons that is used to detect molecular structure.
  • Background information:
    • Protons spin, which produces a magnetic field.
    • Radio waves have a magnetic field.
    • Radio waves are sent onto protons so that the protons experience an external magnetic field.
      • This external magnetic field a
    • The proton spin will either:
      • [Lower Energy]: Align with the external magnetic field
      • [Higher Energy]: Oppose the external magnetic field
    • Resonance Frequency = the difference between the high and lower energy spin states.
    • Chemical Shift = the resonance frequency of the compound compared to the resonance frequency of a reference compound (tetramethylsilane).
      • Units: ppm (parts per million)
  • How it works:
    • NMR pumps radio waves into protons in order to move them to higher energy level spins.
    • When the radio waves are removed, the protons return to their base energy level spin and
    • As the protons return to their base state, a magnetic field change occurs and is recorded as peaks in the spectra.
  • # of peaks for a certain proton = n + 1
    • n: # of hydrogens on the adjacent carbons
  • Area under each peak = # of protons
  • If the proton is near an electron withdrawing group, the proton will be deshielded and experience a larger magnetic field.
    • Thus, these protons show peaks on the left of the spectra; larger chemical shifts
  • If the proton is NOT near an electron withdrawing group, it will be shielded by electrons and experience a smaller magnetic field.
    • Thus, these protons show peaks on the right of the spectra; smaller chemical shifts

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Infrared (IR) Spectroscopy

 

X-axis and Y-axis of spectrum?

What does a valley (dip) in the spectrum mean?

Fingerprint region?

Where do the following occur on the IR spectrum?

  • Functional groups with hydrogens?
  • ..without hydrogen?
  • Carbonyl groups (C=O)?
  • Order of single bonds, double bonds, triple bonds.

Infrared (IR) Spectroscopy

Deals with the way IR light causes bonds with dipoles to vibrate and rotate

  • Slowly changes the frequency of infrared light that is shined on a compound and records the frequencies of light that the compound absorbs.
  • Technique used to determine the functional groups in a compound.
    • Does not determine the structure of the compound.
  • Infrared light is put on a compound, which causes the polar bonds to experience a dipole moment, causing stretching and contracting.
    • Causes intramolecular vibrations and rotations.
    • Bonds compress and bend like a spring, also rotate.
  • X-axis: wavenumber (cm-1 )
    • 1/wavelength
    • Directly proportional to frequency
  • Y-axis: % transmittance
    • % of light that passes through the compound.
    • % of light that is NOT absorbed.
  • A valley (dip) in the spectrum indicates that the compound absorbed infrared light at that wavenumber.
  • Fingerprint region = region of the IR spectrum that is unique for each compound. 
    • It is below 1300 cm-1
  • Functional Groups:
    • w/ hydrogens (C-H, OH, N-H)
      • Anything around 3000 cm-1 
    • without hydrogens 
      • Anything around or below 2000 cm-1
    • Carbonyl groups (C=O) 
      • 1700 cm-1
    • Triple bonds, Double bonds, Single bonds

          (higher wavenumber)         (lower wavenumber)

  • Triple and double bonds vibrate faster, thus they vibrate at higher wavenumbers.

 

 

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Ultraviolet (UV) Spectroscopy

  1. How does it work?
  2. What happens to a conjugated compound when UV light is shined on it?
  3. What range of wavelengths is the UV region?
  4. X-axis of spectrum? Y-axis?
  5. There is a _____ nm increase for each additional conjugated double bond (fill in blank).
  6. There is a ____ nm increase for each additional alkyl group (fill in blank).

Ultraviolet (UV) Spectroscopy

Deals with the way UV light affects in conjugated compounds.

  • UV spectroscopy detects conjugated compounds (double bonds separated by single bonds).
    • It does this by recording the amount of light that a compound absorbs at different wavelengths of light.
    • Tells us how much light the compound absorbs at different wavelengths of light.
  • When UV light (photons) shines on a conjugated compound:
    • Either π-electrons or nonbonding electrons transition to antibonding orbitals.
  • UV region wavelength range: 200-400 nm
  • X-axis: wavelength (nm)
  • Y-axis: absorbance
  • There is a 30-40 nm increase for each additional conjugated double bond in a compound.
    • The more conjugated double bonds, the higher the wavelength of light a compound absorbs.
  • There is a 5 nm increase for each additional alkyl group in a compound.
    • The more alkyl groups, the higher the wavelength of light a compound absorbs.

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Visible region of light

Visible region wavelength range?

What happens to a compounds light absorbance when it undergoes structural changes? Indicators?

Visible Region of Light

  • 400-700 nm wavelengths on the electromagnetic spectrum.
  • Compounds absorb a certain color, and reflect the complementary colors back to your eyes.
    • You only see the object's color as the complementary colors that it reflects.
  • Structural changes can change a compound's light absorbance.
    • Indicators are compounds that are used to display the endpoint of a titration.
    • pH changes cause indicators to change colors, because their light absorbance properties are changed.

 

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Mass Spectrometry

How does it work?

What is it used for?

Parent peak?

Mass Spectrometry

  • Used to determine a compound's molar mass and its molecular formula.
  • In a mass spectrometer, molecules of a sample are bombarded with electrons, causing the molecules to break apart and to ionize
    • It ionizes and fragments molecules in a sample, and then fires the fragments through a magnetic field.
    • The magnetic field separates the ions based on their size.
    • The spread out ions reach a detector, which records their size.
  • Parent peak - the peak furthest to the right which represents the mass of the molecular ion being studied.

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DNA Denaturation

DNA denaturation = the separation of two strands of DNA

  • Occurs when the hydrogen bonds connecting the two strands are disrupted.
  • Due to exposure to high heat, high salt concentration, or pH changes

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Nucleic Acid Hybridization

Nucleic Acid Hybridization (Annealing) = when two complementary single-stranded nucleic acids (DNA or RNA) anneal together

  • Forms double-stranded nucleic acids.
  • Hybridization = Annealing

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DNA methylation (gene regulation)

When genes are methylated, they are turned off

 

 

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Recombinant DNA

Recombinant DNA = artificially made double-stranded DNA composed of two strands that come from different sources.

 

It is DNA that is not found in the original genome.

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Gene cloning

Gene cloning = process of copying a gene

 

  • can be done via:
    • Polymerase Chain Reaction (PCR)
    • Inserting the gene into a plasmid, then put it in a bacteria.
      • The bacteria will duplicate via binary fission until you have many of bacteria with the gene.
      • Then you can break the bacterial cells and collect the gene clones.

 

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Complementary DNA (cDNA)

How is it made?

Does it have introns?

Complementary DNA (cDNA) = double-stranded DNA synthesized from a single-stranded mRNA via reverse transcription.

  • Because it comes from mature mRNA, it has no introns.
    • mRNA loses its introns during post-transcriptional processing.

Original DNA ⇒ mRNA ⇒ cDNA (single stranded) ⇒ cDNA (double stranded)

  • Reverse Transcriptase is the enzyme that catalyzes the reverse transcription reaction
    • mRNA ⇒ DNA 

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Polymerase Chain Reaction (PCR)

How does it work (steps)?

What is needed to do this?

Polymerase Chain Reaction (PCR)

  • Technique used to clone DNA at an exponential rate
    • 2x = amount of DNA obtained
    • x: number of PCR cycles done.
  • How it works:
    1. Double-stranded dNA is placed in a mixture containing many copies of DNA primers for each strand.
    2. Mixture is heated to denature DNA into two separate strands.
    3. Mixture is cooled so the primers anneal (hybridize) to their complementary ends on the DNA strands.
    4. Taq polymerase and many nucleotides are added to the mixture.
    5. The mixture is heated, which activates taq polymerase, causing it to replicate the DNA.
    6. These heating/cooling cycles are repeated to make many copies of the DNA.
  • Needs:
    • Double-stranded DNA of interest
    • taq polymerase (heat resistant)
    • DNA primers
    • Nucleotides

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Genetic Knockout Organism

Genetic Knockout Organism - organism that has a certain gene removed from its DNA.

 

 

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Human gene therapy

Gene Therapy - a defective allele of a gene is replaced with the wildtype (normal) allele.

 

can sometimes be successful.

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Biometry

Biometry - the use of statistics to understand biological data

 

Example: can be used to study population genetics on a large scale

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Restriction Enzymes

 

What makes them? Defense function?

Can they read both forward and backward?

Restriction Enzymes - enzymes that cleave nucleic acids (DNA/RNA) only at certain nucleotide sequences

 

  • They are made by bacteria, which use restriction enzymes to destroy viral DNA for protection.
  • Restriction enzymes can read the restriction sequences forward and backward.
  • Two DNA fragments that are cleaved by the same restriction enzymes can easily be connected.

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•NMR does not detect functional groups.

•It detects the arrangement of hydrogen and carbon atoms in a compound

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•Find the # of unique carbons in each compound.

•Do not repeat the same type of carbon.

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is precipitation exothermic or endothermic?

Precipitation is exothermic

- ΔH

 

 

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