Separating Mixtures Flashcards
Unit 2
Define chromatography
Separation of the components of a mixture dissolved in the mobile phase carrying it through the stationary phase.
Why might we want to separate a mixture?
Identify, purify and quantify substances present.
what do we call the 2 phases used in chromatography?
Mobile and stationary
why do substance in a mixture move to different distances?
They have different affinities for the 2 phases
Greater affinity for which phase means substance move more slowly?
Greater affinity for the stationary
Why might a mixture of solvent be used ?
To separate substances that are equally soluble in 1 solvent
Define adsorption
when a substance (e.g.gas, liquid or solute)binds or attaches to another (usually solid)
Define polar/hydrophilic
a polar substance will dissolve in or mix with water.
Define adsorbent
describes the stationary phase in chromatography because substances become adsorbed to it during separation
What is one advantage of paper chromatography over other types?
it is cheaper
In paper chromatography, what stationary phase?
water bound to the paper.
In paper chromatography , what is the mobile phase?
A non-polar solvent or mixture of solvents
What do we call the line drawn at the bottom of the where the solvent has travelled to?
Origin
what is the name of the line at the top of the paper where the solvent has travelled to?
solvent front
what do we call the paper that has the separated substances on it at when chromatography has been carried out?
chromatogram
what does Rf value mean?
Retention factor
how is Rf value calculated?
Rf=distance travelled by substance/distance travelled by solvent
how many decimal places are Rf values expressed to?
2 d.p
when chromatography is carried out using a stardoms procedure, what can Rf values be compared to identify substances in a mixture?
Published literature values
what do we mean by standard procedure?
- same solvent
- same stationary phase
- same run time
- same temperature
Another way of identifying substances in a mixture is by using reference standards; what are reference standards.
- samples of pure substances (standards) applied the origin next to the mixture
- to compare distance travelled by spots
substances or processes that make spots on a chromatogram visible?
locating agents
Give one example of a non-destructive locating agent
fluorescent substances can be seen with UV light
Give examples of other locating agents
ninhydrin (shows amino acids),iodine (shows starch)
what are the advantages of TLC over paper chromatography
- faster run time
- better separation of substances
- choice of absorbents
- TLC plate self-supporting/ easy to manipulate
- TLC more durable
what’re the disadvantages of Bothe paper chromatography and TLC?
- Don’t give positive identification of substances
- Qualitative not Quantitative results
What is TLC?
Thin layer chromatography
In TLC, what is the stationary phase?
silica gel, powder cellulose or alumina/aluminium oxide attached to glass or plastics.
in TLC, what is the mobile phase?
`a non-polar solvent or mixture of solvents
On what basis does gel electrophoresis separate DNA fragments?
gel electrophoresis separates DNA fragments based on the size of the fragments
List the uses of gel electrophoresis and genetic fingerprinting
forensic science paternity testing genetic disease diagnosis pathogenic disease diagnosis tracing ancestry determining phylogenetic relationships between organisms
What charge do DNA molecules have?
Negative
When DNA fragments are placed in an electrical field, which electrode are they attracted to?
the positive electrode (anode)
What is the gel in gel electrophoresis made from?
agarose
Which DNA fragments can move through the pores in the gel matrix more easily (and can therefore move further in a set time)?
Shorter fragments
What is the name of the enzymes that are used to cut DNA molecules into fragments before gel electrophoresis?
restriction enzymes
What are the DNA fragments placed into at one end of the gel?
Wells
What is added to complete the circuit?
Buffer solution
Which electrode is placed near the wells?
the negatively charged electrode(cathode) is placed near the wells
What is important about the electrical current applied?
direct current
What is used to visualise the DNA fragments?
dye or radioactivity
What unit of measurement is used for DNA fragments?
base pairs (bp)
Why is a ‘size reference standard’ mix of DNA fragments used in one of the lanes in gel electrophoresis?
to estimate the size of the DNA fragments in the other lanes
What process is used to make a permanent record of the gel?
Southern blotting
What do we call the position or pattern of the DNA fragments in the gel?
DNA bands or banding pattern
When looking at genetic fingerprints created by gel electrophoresis, how do we know that 2 individuals are closely related?
they have (more) bands (DNA fragments) in common
What does PCR stand for?
polymerase chain reaction
What is PCR used for?
to make millions of copies of a target piece of DNA
What 3 things must be added to the DNA for PCR to be carried out?
nucleotides, primers, & polymerase
How are DNA strands separated in PCR?
heat to 95oC for 2 minutes
To start the replication process, what must attach or anneal to the DNA?
Primers
Which enzyme is needed to form the new DNA copies?
Taq DNA polymerase
Which organism does the DNA polymerase come from?
Thermus aquaticus bacteria
What is the advantage of using Taq DNA polymerase?
It doesn’t denature at high temperatures (needed to separate DNA strands)
What is the formula for calculating the number of DNA molecules after a certain number of PCR cycles?
2n (where n = the number of cycles)
Why is it necessary to cool the mixture after heating to separate the DNA strands?
to allow the primers to attach/anneal
