Laboratory Techniques Flashcards by Jeet Das

Separations

Lab techniques that use intermolecular forces to separate mixture into its component parts

Solubility, melting point, boiling point

Includes extraction, distillation, crystallization, chromatography

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Extraction

Separation based on solubility

Two immiscible phases (aqueous and less dense organic)

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Distillation

Separation based on boiling points

Lower boiling point boils off first

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Fractional distillation

More precise distillation for liquids w/ similar boiling points

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Crystillization

Separate based on purity

Form crystals more easily using pure substances than impure (inefficient)

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Chromatography

Purify compound from mixture and identify ratio of compounds in mixture

Mobile phase and stationary phase

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Column chromatography

Solution is poured down column w/ solid phase (glass beads)

More polar compounds travel down more slowly

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High pressure liquid chromatography (HPLC)

Puts system under high pressure instead of gravity

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Paper chromatography

Spot portion of sample on paper => place in nonpolar solvent and solvent moves up via capillary action, dissolving sample as it passes

Separates more polar from less polar contents (more polar moves slower)

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Rf factor

Divide distance traveled for component by distance traveled by solvent

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Thin-layer chromatography

Coated glass/plastic plate instead of paper

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Gas-liquid chromatography

Liquid phase is stationary phase

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Size-exclusion chromatography

Separated by size/molecular weight

Gel filtration

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Ion-exchange chromatography

Separated based on net surface charge using anionic/cationic exchangers

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Affinity chromatography

Highly specific interactions slow down select molecules

Receptor-ligand, enzyme-substrate, antigen-antibody

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Gel electrophoresis

Separate mixtures of nucleic acids/proteins based on size and charge

Negative nucleic acids migrate, larger particles move slowly

Proteins are denatured first b/c too large

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Isoelectric points

Point at which protein has no net charge

In gel electrophoresis, (-) charged protein migrates until it loses charge => bands show up at isoelectric points

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Southern blotting

Identify target fragments of known DNA sequence in large population of DNA

Uses gel electrophoresis and hot probe

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Northern blot

Identify RNA fragments

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Western blot

Detect particular protein in mixture of proteins

Uses antibodies (primary and secondary)

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Primary antibody

Specific to protein

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Secondary antibody

Recognizes primary antibody and marks it for visualization

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Separation of enantiomers

Differences in crystallization, stereospecific enzymes, convert to diastereomers w/ different properties

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Chiral resolution

Separation of enantiomers from racemic mixture

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Spectroscopy

Interaction b/w matter and electromagnetic light

Nuclear Magnetic Resonance (NMR) spectroscopy

Interaction b/w atomic nuclei and radio waves Used to study hydrogen or carbon nuclei

Nuclear spin (NMR)

Creates magnetic field around nucleus that can align with/against external magnetic field Magnetic spin of H/C nuclei Only exists in nuclei w/ odd atomic/mass numbers

Energy of photon

∆E = hf ``` h = Planck's constant f = frequency ```

Chemical shift (NMR)

Same for two hydrogens with indistinguishable positions by NMR Difference b/w resonance frequencies of chemically shifted H and reference compound

Splitting of peaks (NMR)

Caused by neighboring H that are not chemically equivalent AKA spin-spin splitting

IR spectroscopy

Infrared radiation => polar bonds in compound stretch and contract (Intermolecular vibrations and rotation) Bonds w/ dipoles vibrate/rotate

C=O peak (IR)

Sharp dip around 1700

O-H peak (IR)

Broad dip around 3200-3600

Atoms with greater mass resonate at ______ (IR)

Lower frequencies

Stiffer bonds resonate at _____ (IR)

Higher frequencies

Fingerprint region

Distinguish one compound from another 600-1400cm^01

Ultraviolet region of EM spectrum

Between 200 and 400 nm

UV spectroscopy

Detects conjugated systems (double bonds separated by one single bond)

If compound has 8+ double bonds... (UV)

Absorbance moves into visible region

Beta-carotene

Precursor of Vitamin A w/ 11 conjugated double bonds

Complementary color

Green => Red Blue => Orange Yellow => Purple

Mass spectrometry

Determines molecular weight and sometimes formula Bombard sample w/ electrons => break apart and ionize

Molecular ion

Cation w/ largest mass and one less electron than original molecule

Base peak

Largest peak

Parent peak

Made by molecular ions that did not fragment (farthest right of spectrum)

Denature dsDNA

Heat, salt, high pH

Nucleic acid hybridization

Separated strands spontaneously associate w/ original parter or any complementary sequence

DNA methylations

Bacteria protect own DNA from restriction enzymes by adding CH3 (methyl) group

Restriction enzymes

Digest/cut nucleic acid at certain sequences along chain (restriction sites)

Restriction sites

Palindromic sequences 4-6 nucleotides long

Sticky ends

Complementary single stranded ends

Recombinant DNA

Two DNA fragments cleaved by same endonuclease (restriction enzyme) joined together regardless of origins

Gene cloning

Make many copies of genes

Vector

Engineered plasmid to serve a purpose Usually places recombinant DNA w/in bacterial genome

Complementary DNA (cDNA)

Product of reverse transcribing mRNA produced by DNA using reverse transcriptase Has no introns

Polymerase chain reaction

Heat (denature) Cool (anneal primers) Add polymerase + nucleotides Heat (activate polymerase) Exponential increase in DNA amount

Formula for PCR

x2^n ``` x = starting amount n = number of cycles ```

Sanger/chain-termination method

Dideoxynucleotides incorporate into growing DNA but terminate chain

Determining gene expression

``` Evolution Knock-out RNAi Overexpress (increase promoter strength) Introduce into new cell line/organism ```

RNA interference

Small pieces of RNA bind to mRNA => prevent translation and mark for degradation Reduces gene expression

Restriction fragment length polymorphism (RFLP)

Identifies individuals by fragmenting DNA w/ endonucleases and southern blotting (DNA fingerprint)

Human gene therapy

Genetic manipulation of affected individual's DNA Can replace defective allele w/ wildtype

How do you extract the undesired by-product?

Deprotonate/protonate it so that it is in ionic/charged form and will extract into the aqueous layer