- Separations divide mixtures into their components based on intermolecular forces and can be used for purification, identification, or collection.
- Spectroscopy and spectrometry provide structural information that can be used to identify compounds.
- Genetic techniques have many applications, including the isolation/identification of genes and the manipulation of DNA on the molecular level.
Separations:
Extraction - Separation technique based on solubility. Involves an aqueous layer and a less dense organic layer.
-Main idea: "like dissolves like"
-Weak acids protonate strong bases and strong bases protonate weak bases.
-Weak bases deprotonate strong acids and strong bases deprotonate weak acids.
All additions stated above are done in the order stated, as shown in the figure below.
Distillation - Technique used to separate compounds that have significantly different boiling points.
-At least 20°C difference
-The lower boiling point liquid is boiled first and then condensed to go to the side tube.
-Fractional Distillation can be used for two liquids that are closer than a 20°C difference in boiling point.
Crystallization - Is based on the principle that pure substances form crystals more easily than impure substances.
-Impure substances have higher freezing and melting points.
-Mostly an exothermic process
Chromatography - Can be used to purify a compound from a mixture and/or to identify the ratio of compounds in a mixture.
-Typically, the stationary phase is polar, causing polar substances to exude more slowly.
Column chromatography - Solution containing the mixture is dripped down a column containing the solid phase. The more polar compounds in the mixture travel more slowly down the column. Gravity drives the movement down.
Paper chromatography - One end of paper is placed in a non-polar solvent. The solvent moves up the paper via capillary action and dissolves the sample as it passes over it.
-Rf factor = Distance traveled/Total Distance
Thin-layer chromatography - Similar to paper, except that a coated glass or plastic plate is used instead of paper.
***Column and thin layer chromatography separate compounds by exploiting differences in the polarity of their molecules.
Gas-liquid chromatography - Liquid phase is the stationary phase.
Size-exclusion chromatography - Molecules are separated by their size and sometimes molecular weight,
Ion-exchange chromatography - Molecules are separated based on their net surface charge.
Affinity chromatography - Uses highly specific interactions to slow down select molecules, rather than simply separating out all molecules that have a particular property.
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Mixtures of nucleic acids or mixtures of proteins can be separated based on size and charge through gel electrophoresis.
-Since the agarose gel used for separation form a porous matrix, smaller pieces move more easily through those pores, and nucleic acids migrate through the gel in response to electric field, since nucleic acids are negative.
-Proteins migrate at a rate proportional to their charge and length.
-Proteins can be separated via gel electrophoresis based on their isoelectric points.
Blotting - Is a technique by which molecules are transferred from the gel onto a membrane, maintaining the same spatial relationship of bands based on size.
Southern blotting - Is a technique used to identify target fragments of a known DNA sequence in a large population of DNA.
- Chop up some DNA
- Use an electric field (gel electrophoresis) to spread out pieces according to size
- Blot it onto a membrane
- Add a radioactive prove made from DNA or RNA
- Visualize
Northern blotting - Is just like Southern blotting, but identifies RNA fragments, not DNA fragments.
Western blotting - Is used to detect a particular protein in a mixture of proteins.
-Antibodies are used to spot the protein.
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Separation of enantiomers from a racemic mixture (chiral resolution), is a challenge because enantiomers have almost exactly the same physical and chemical properties. There are three ways to resolve enantiomers.
- Differences in crystallization of the enantiomers. The direct visualization of the crystals can be used to separate enantiomers.
- Stereospecific enzymes added to a racemic mixture will react to only one enantiomer in the mixture, creating a compound that can be separated using separation techniques.
- Enantiomers in a racemic mixture can be converted into diastereomers, which have different physical and chemical properties that can be used for separation.
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Spectroscopy/Spectrometry:
Spectroscopy/Spectrometry:
Spectroscopy:
Spectroscopy is the study of the interaction between matter and electromagnetic radiation (light).
Splitting of a peak into several smaller peaks is caused by neighboring hydrogens that are not chemically equivalent.
-The number of peaks due to splitting for a group of chemically equivalent hydrogens is given by the simple formula n+1, where n is the number of neighboring hydrogens that are not chemically equivalent.
Interpreting proton NMR spectroscopy:
- Identify chemically equivalent hydrogens.
- Identify and count neighboring hydrogens that are not chemically equivalent. Use n+1 to determine the number the peaks created by splitting for the chemically equivalent hydrogens.
- If necessary, identify electrons withdrawing/donating groups near the chemically equivalent hydrogens. Withdrawing groups will move the signal to the left, and donating groups will move the signal to the right.
Aldehydes = 9.5
COOHs = 10-12
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IR spectroscopy - Uses molecular dipoles to find information about functional groups.
-When exposed to radiation in the infrared region of the electromagnetic spectrum, the polar bonds within a compound stretch and contract, causing intramolecular vibrations and rotations.
Many of the complex vibrations that distinguish one compound from a similar compound are found in the 600 to 1400 cm-1 region, called the fingerprint region.
UV Spectroscopy - Detects conjugated systems by comparing the intensities of two beams of light from the same monochromatic light source. One beam is shone through a sample cell and the other is shone through a reference cell.
-30 to 40nm increase for each additional conjugated double bond, and a 5nm increase for each additional alkyl group.
-If a compound has 8 or more double bonds, its absorbance moves into the visible region of the electromagnetic spectrum.
All in all, NMR deals with the magnetic spin of hydrogen or carbon; IR deals with the how IR light causes bonds with dipoles to vibrate and rotate; and UV deals with how UV light affects electrons in conjugated systems.
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*Spectroscopy studies interactions between matter and electromagnetic radiation.
*Spectrometry studies the interactions between matter and energy sources other than electromagnetic radiation.
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Mass spectrometry - Used to determine a compound's molecular weight, and in the case of high resolution mass spectrometry, its molecular formula.
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Genetic Techniques:
Three types of genetic laboratory techniques:
-30 to 40nm increase for each additional conjugated double bond, and a 5nm increase for each additional alkyl group.
-If a compound has 8 or more double bonds, its absorbance moves into the visible region of the electromagnetic spectrum.
All in all, NMR deals with the magnetic spin of hydrogen or carbon; IR deals with the how IR light causes bonds with dipoles to vibrate and rotate; and UV deals with how UV light affects electrons in conjugated systems.
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*Spectroscopy studies interactions between matter and electromagnetic radiation.
*Spectrometry studies the interactions between matter and energy sources other than electromagnetic radiation.
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Mass spectrometry - Used to determine a compound's molecular weight, and in the case of high resolution mass spectrometry, its molecular formula.
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Genetic Techniques:
Three types of genetic laboratory techniques:
- Nucleic acid manipulation - DNA and RNA can be manipulated on a macro or micro level. Techniques can be used to join two strands together, pry two strands apart, chop one piece into smaller pieces, or join smaller pieces into a alarger piece.
- Recombination and cloning - Many copies of the same piece of genetic material can be created and put in new places or otherwise manipulated.
- Sequencing and function - Techniques can be used to determine the exact nucleotide sequence of a piece of DNA or RNA. Additionally, a piece of nucleic acid that codes for a protein can be used to determine the function of that gene or protein.
Nucleic acid manipulation:
*Denaturing is going from 2 strands to 1 strand. Hybridization is going from 1 strand to 2 strands.
Recombination and cloning:
-Restriction enzymes (aka restriction endonucleases) are used to cut DNA into fragments. Bacteria protect their own DNA from these enzymes through DNA methylation.
-Recombinant DNA is DNA that has been artificially recombined. Scientists can 'cut' and digest multiple pieces of DNA with the same restriction enzyme and then 'paste' the pieces together.
As shown in the figure above, to make a DNA library, take a DNA fragment, use a vector to insert it into a bacterium, and reproduce that bacterium like crazy. Now you have clones of bacteria with the DNA fragment.
cDNA is just DNA reverse transcribed from mRNA. The great thing about cDNA is that it lacks the introns that would normally be found in eukaryotic DNA.
PCR (Polymerase chain reaction) - Is a much faster way to clone.
Sequencing and function:
DNA Sequencing: Sanger method:
Gene Chip Method:
RFLP (Restriction fragment length polymorphism) - Identifies individuals, rather than identifying specific genes.
Human gene therapy is used by replacing defective alleles with wildtypes, or correctly functioning alleles.
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