Lab techniques Flashcards
in extraction it is better to seqeentual steps compared to a single step.
To seperate multiple compenonts, the first extration uses a weak acid or base to extract the strong elements. THEN a strong acid or base is used to extract the remaining weak componets.
If the reverse order was used, a strong acid or base in the first separation would react with all strong or base in the first separation would react with - all strong and weak - complenets such that would be extracted all at once from the organic into the aq layer rather than being sequentially seperated from one another.
Boiling point in inversely related to vapor pressure
if you increase boiling point, you decrease vapor pressure
example if you add salt into water, you decrease the vapor pressure and as a result you increase the boiling pt
distillation - seperation via boiling points.
lower boiling point will evaporate first.
Name and define the 3 types of distillation
distillation
1. simple distillation
- Vacuum distillation
- fractional distillation
Name and define the 3 types of distillation
distillation
1. simple distillation - boiling points are at least 25 degrees away from each other.
- Vacuum distillation - if the boiling point is greater than 150, the vacuum will decrease the boiling point.
- fractional distillation - if the 2 solvent’s boiling point is within 25 degrees from each other.
Crystallization - is based on the principle that pure substances will form crtysals more easily than impure substances
pure crystals will have a higher melting point than impure crystals because impure crystals wil mess with the intermolecular forces and not allow for the strong bonds to occur like in the pure crystal.
recrystallization is a organic chem techniwue to obtain a purer crystal.
pure crystals wil only melt at higher temperatures.
if you want to get something of out solution –> crystal, you want to lower the temperature so that the crystal can from and then want the solubility at the particular temperature to be low so the crystal can come out of solution.
is crystyallation an exothermic or endothermic proces?
you are forming bonds, therefore it is a exothermic process
Chromatography - can be used to PURIFY compounds from a mixture and /or to idenify the ratio of compound in a mixture
solution = mobile phase
stationary phase = what adsorb( bind ) to seperate
column chromatography - solution mixture travel through a colum containing beads
more similar affinity to bead ( slower moving)
usually gravity gets the trick done.
the faster eluted molecules have very little affinity to the beads
column chromatography - solution mixture travel through a colum containing beads
more similar affinity to bead ( slower moving)
usually gravity gets the trick done.
the faster eluted molecules have very little affinity to the beads
stationary phase : zpolar gel or powder
mobile phase: nonpolar solvent
purpose: serpare a sample into components
HIgh pressure liquid chromatography ( HPLC)
similar to column chromatography - but puts the system under high pressure
mobile phase: nonpolar solvent
stationary phase: crushed metal or polymer
purpose: seperate vaporizable compounds
Paper/thin Chromatography = polar paper ( stationary phase )
mobile phase = nonpolar - travels via capillary action
purpose: idenify sample
more polar move less since it likes the stationary phase paper which is polar
less polar - moves higher on the paper
Rf factor can be calculated fro each compoint by dividing the distance traveled by the distance traveled by the solvent,.
Nonpolar compunds have a rF factor close to 1 and polar compomounds have lower rf factor values.
gas-liquid chromatography - what is the stationary and the mobile phase??
- liquid is stationary phase
- gas is the mobile phase
mixture dissolved in a heat - carrier gas ( He or N2) + passed over the liquid phase bound to a column.
compound in mixture equilibriates - with liquid phase at different rate and pass through an exit port as individual compound.
gas-liquid chromatography - what is the stationary and the mobile phase??
- liquid is stationary phase
- gas is the mobile phase ( inert gas)
mixture dissolved in a heat - carrier gas ( He or N2) + passed over the liquid phase bound to a column.
compound in mixture equilibriates - with liquid phase at different rate and pass through an exit port as individual compound.
Purpose: to seperate vaporazable compounds
size - exclusion chromatography
mobile phase : nonpolar solvent
stationary phase: polar, porous beads
molecular seperated by size ( sometimes via molecular weight )
smaller molecules get struck on the bead ( stationary phase)
purpose: serperate components by charge
ion-exchnage chromatography
mobile phase: nonpolar solvent
stationary phase: charged beads in column
prupose: serpate components by charge
molecule is seperated based on net surface charge.
“ cationic/anionic” exchangers that slow down movment of charged particle
Seperation of peptides and protien can be done how?
- size exlusion chromatography
- ion-exchange chromatography
- affinity chromatography
Affinity Chromatrography
mobile phase : nonpolar solvent
stationary phase: beads coated with antibody or receptor for a target molecule
purpose: purify a molecule ( usually a protien of interest )
highly specidin interactions to slow down selected molecule rather than simply seperating out all moelcules that have particular properties
example: receptor-ligand
enzyme-substate
antigen-antibody interaction
reverse phase chromatrography
mobile phase: polar solvent
stationary phase: nonpolar card
purpose : to idenify sample:;
gel electrophoresis - define its uses
can be used to serparate DNA? RNA/ protien fragements by charge/ size and needs a electric field to be applied
nuclelic acid is negatively charged.
larger molecules move more slowly in the pores on the gel
SDS -PAGE ( polyacrylamide gel electrophoresis) - used for protien.
but the protien has been denatured
Southern Blot - typicaly used to idenify frag of DNA
recipe for Southern blotting
- Chop up DNA
- use electric field ( gel - electrophoresis) to seperate out pieces according to size
- Sourthern BLOT on membrane ( nitrocellulose)
- add radioactive probe madde from ( RNA or DNA) which is complementary to target fragment + will hybridize –> mark fragment
- visualize ( typically it fluorseces )
Southern Blot - typicaly used to idenify frag of DNA
recipe for Southern blotting
- Chop up DNA
- use electric field ( gel - electrophoresis) to seperate out pieces according to size
- Sourthern BLOT on membrane ( nitrocellulose)
- add radioactive probe madde from ( DNA) which is complementary to target fragment + will hybridize –> mark fragment
- visualize ( typically it fluorseces )
Northern blot -> typically used to idenidty RNA
recipe for Northern blotting
- Chop up RNA
- use electric field ( gel - electrophoresis) to seperate out pieces according to size
- Northern BLOT on membrane ( nitrocellulose)
- add radioactive probe made from ( RNA) which is complementary to target fragment + will hybridize –> mark fragment
- visualize ( typically it fluorseces )
Northern blot -> typically used to idenidty RNA
recipe for Northern blotting
- Chop up RNA
- use electric field ( gel - electrophoresis) to seperate out pieces according to size
- Northern BLOT on membrane ( nitrocellulose)
- add radioactive probe made from ( RNA) which is complementary to target fragment + will hybridize –> mark fragment
- visualize ( typically it fluorseces )
Western blot ( -> typically used to identify protien
it uses antibodies to do so.
Recipe for western Blotting
- Chop up the Protien
- Use gel electrophoresis to seperate out the pieces according to size.
- transfer the fragments onto a nitrocellulose membrane
- place ( 1*) primary antibody on to the protien of interest on the membrane.
- 2* ( Seconary antibody - enzyme conjugate will bind to the 1* antibody and marks it with the enzyme ( for visualization ) –> enzyme will then catalyze reaction to produce colored/fluroscent or radtioactive reaction for visualization.
Enantiomers - since they have the same physical and chemical properties there are 3 ways to seperate them out. name them.
- use difference in crystallization of the enantiomer - direct visualization of the crystal can be used to seperate enantiomers
- add sterospecific enzyme that can be added to a racemic mixture and it will react with only one enantiomer . now this will create a compund that can be seperated by seperation techniques. - once the alterd enantiomer is removed, the reaction can be reversed to produce the pure enviroment
- enantiomers can be convered into dissteromers - which have different phsycail and chemical roperties that can be used for seperation.
Enantiomers - since they have the same physical and chemical properties there are 3 ways to seperate them out. name them.
- use difference in crystallization of the enantiomer - direct visualization of the crystal can be used to seperate enantiomers
- add sterospecific enzyme that can be added to a racemic mixture and it will react with only one enantiomer . now this will create a compund that can be seperated by seperation techniques. - once the alterd enantiomer is removed, the reaction can be reversed to produce the pure enviroment
- enantiomers can be convered into dissteromers - which have different phsycail and chemical properties that can be used for seperation.
Enantiomers - since they have the same physical and chemical properties there are 3 ways to seperate them out. name them.
- use difference in crystallization of the enantiomer - direct visualization of the crystal can be used to seperate enantiomers
- add sterospecific enzyme that can be added to a racemic mixture and it will react with only one enantiomer . now this will create a compund that can be seperated by seperation techniques. - once the alterd enantiomer is removed, the reaction can be reversed to produce the pure enviroment
- enantiomers can be convered into dissteromers - which have different phsycail and chemical properties that can be used for seperation.
distilation is more effective when it is done slowly, as this gives the compound with a lower boiling point time to completely boil off
distillation - while it is a generally effective method - it cannot completely seperate two compounds.
distilation is more effective when it is done slowly, as this gives the compound with a lower boiling point time to completely boil off
distillation - while it is a generally effective method - it cannot completely seperate two compounds.
name the different types of spectroscopy
- NMR ( nuclear amagnetic resonance )
- Infrared ( IR)
- Ultraviolet - visible ( UV-VIS)
NMR - radiowaves - used for arragement of carbon and hydrogen bonds
upfield - sheilded with electrons
downfield - deshielded with electrons - EWG - more acidid protons would be here.
Infrared ( IR) - infrared light - presnce of funciton gorup
via vibrating the molecule.
if there is no dipole aka if something like nitrogen gas undergo IR, there will be no spectrum since N2 gas expereince no dipole/vibration
UV-VIS ( ultraviolet - visible light ) - presence of the conjugated PI systems
this is why you can see color. it is part of the ultraviolet and visible spectrum.
NMR - radiowaves - used for arragement of carbon and hydrogen bonds
upfield - sheilded with electrons
downfield - deshielded with electrons - EWG - more acidid protons would be here.
magnetic spin of ODD numbered atoms ( hyfrogen or carbon - 13 nuclei
NMR - radiowaves - used for arragement of carbon and hydrogen bonds
splitting n+1 where N is the number of hydrogens that are neighboring not chemically equivalent.
upfield - sheilded with electrons - EDG
downfield - deshielded with electrons - EWG - more acidid protons would be here.
magnetic spin of ODD numbered atoms ( hyfrogen or carbon - 13 nuclei
NMR - radiowaves - used for arragement of carbon and hydrogen bonds
splitting n+1 where N is the number of hydrogens that are neighboring not chemically equivalent.
upfield - sheilded with electrons - EDG
downfield - deshielded with electrons - EWG - more acidid protons would be here.
magnetic spin of ODD numbered atoms ( hyfrogen or carbon - 13 nuclei
if you are dealing with the carbon NMR, do the same thing as hydrogen but IGNORE THE SPLITTING.
Infrared ( IR) - infrared light - presnce of funciton gorup
via vibrating the molecule.
if there is no dipole aka if something like nitrogen gas undergo IR, there will be no spectrum since N2 gas expereince no dipole/vibration
UV-VIS ( ultraviolet - visible light ) - presence of the conjugated PI systems
this is why you can see color. it is part of the ultraviolet and visible spectrum.
aldehyde shift on NMR :
9.5 ppm
Carboxylic acid shift on NMR
10-12 ppm.
IR spectrum values to know.
O-H
Carbonyl ( C=O)
O-H - wide 3200-3600 cm^-1
C=O narrow sharp dip around 1700 cm^-1
spectrometry
the study of interactions between matter and energy sources other than electromagnetic radiation.
Mass spectrometry - is used to determine the compounds molecular weight ( or its formula )
- a molecule can be bombarded with electrons in order to break them apart and ionize.
Largest ion = molecular ion = orginal molecule without an electron
Example if the original molecule was methane ( CH4) the molecular ion will be (CH4+)
the particules move along a magnetic path - mass/charge ratio ( m/z)
parent peak = made by the molecular ion ( it does not fragment )
base peak = largest peak and all of the abundance percentages are made based on the base peak
for C-13 NMR, count the number of unique Carbon Groups and note for symmetry in the molecule.
note for carbon , there is no N+1 splitting.
what factors can denature DNA?
- increased salt concentration
- increased pH
- increased tempertature
Temperature at which double strand –> single strand (Tm aka melting temperature )
as the G-C content increases, the Tm increases as well.
Hybridization
technique that alows scientists to identidy nucleotide seqeunces by binding a known sequence with an unknown sequence.
Hybridization
technique that alows scientists to identify nucleotide seqeunces by binding a known sequence with an unknown sequence.
DNA-DNA , DNA- RNA, RNA - RNA
restriction enzyme ( aka restriction endonucleases) do what?
- digest( cut) nucleic acid only at certain nucleotide sequences along the chain.
typically the restriection site is palindeomic of 4-6 nucelotides long. –> cleaved unevently - leaving sticky ends –> leads to the making of recombinant DNA.
The bacteria will uptake extra DNA from the enviroment via which method?
- transformation
The bacteria will uptake extra DNA from the enviroment via which method?
- transformation
What are the components and the steps of PCR
- DNA
- Primer
- Taq polymerase ( Heat resistant polymerase )
steps:
1. denature by increase temperature
2. decrease temperature and anneal the primers
3. use the polymerase and make the new DNA strands
4. Rinse and repeat
number of DNA = 2^n where n=number of cycles.
What are the components and the steps of PCR
- DNA
- Primer
- Taq polymerase ( Heat resistant polymerase )
steps:
1. denature by increase temperature
2. decrease temperature and anneal the primers
3. use the polymerase and make the new DNA strands
4. Rinse and repeat
number of DNA = 2^n where n=number of cycles.
Sanger method ( Chain termination method ) - desribe it
from its name it terminates the chain with ddNTP ( dideoxynucleotide) it sends the sequence since there is no place for the phosphodiester bond to be made.
Sanger method ( Chain termination method ) - describe
from its name it terminates the chain with ddNTP ( dideoxynucleotide) it sends the sequence since there is no place for the phosphodiester bond to be made.
DNA is read 3’ to 5’ and therefore synthesized 5’ to 3’
so the smallest piece synthesized will migrate all the way down as the smallest piece in the gel electrophoresis.
This method helps figure out the sequence of the DNA of interest.
Analzying gene expression - requires measuring the gene’s downstream mRNA or protiten product
need 2 types of cells. ( before and after)
isolate RNA, reverse transcrpate wit labeled nucleotide
if downreulated - RED
if upregulated =- green
if no change to gene expression = yelow
RNA interfenerece = incomplete loss of function
place small pieces of RNA to bind with mRNA to preent translation from occuuring. and /or marking it for degration
overexpression - a way to determine gene function
increase the strength o the promoter before the gene and therefore it increases the epxression of that gene.
expresion of gene in a new cell line * - way to determine gene function
- gene that codes fro gren fluroscent protien in jelly fish can be placed in organism of an other speices –> produce that protien and glow protoen
Restriction Fragment Length polymorphish ( RFLP)
and single nucleotide polymorphism)
actlike fingerpront for an individual’s genome
Transgenic mice define
mice that contain genes that are transfeered from another species
- it does not override the organizsm’s natural allele /genes
- it is imposible to contorl the number of genes entered, therfore it is good to study the fominant because they dont know if 1 or 2 copies are inserted into the genome.
