Liquid Chromatography 2

Question 1

What are the types of liquid chromatography

Answer 1

Chiral separations

Ion chromatography

Size exclusion

Affinity

Question 2

What are chiral separations

Answer 2

There are chiral molecules at the surface of the stationary phase

The analytes R/S enantiomers have diff interaction with the stationary phase

This causes separation of the R/S enantiomers of the analyte

Question 3

What is derivitization

Answer 3

It helps to enhance the chiral interaction of the stationary phase with the analyte

adds a chromophore to help with UV vis detection

Question 4

What is ion exchange chromatography

Answer 4

The stationary phase has a resin with covalently bound groups that are either acidic or basic

These columns have a max capacity which is determined by the number of binding sites that the analyte binds to (ex. Ca2+ has two binding sites, binds tighter to resin)

The separation depends on the affinity of the analyte for the exchange sites and the porosity of the resin.

Question 5

In ion exachange chromatograph what effect does the acidic and basic groups in the stationary phase have

What effect does the porosity of the resin have

Answer 5

More acidic groups (negative charge) will exhange more with cations

More basic (more positive) will exchange more with anions

The ions passing through the resin have a hydrated radius (solvent around the molecule), if smaller pores of the resin smaller ion penetrated better and are more retained

The smaller

Question 6

What does the order of retention in ion exachange chromatography depend on

Answer 6

Charge (higher charge (2+), more retained, better binding)

Size (smaller hydrated radius, better penetration into resin, retained more)

Polarizabilty (more polarizable, more retained)

Question 7

What are gradient Elutions in ion exchange chromatography

Answer 7

Changing the ph of the eluent changes the binding of the analytes based on their isoelectric points

When the molecule is neutral, it’s binding is reduced and this depend on the ph of what it’s in

Question 8

For cation exchange resins (more acidic) how is elution encouraged

Answer 8

Increasing ph encourages elution (less binding)

Question 9

For anion exchange resins (more basic) how is elution encouraged

Answer 9

Decreasing ph makes less binding

Question 10

What are the detectors used in ion exchange chromatography

Answer 10

They’re conductivity based, and the RF for each ion is detected

UV vis and Charge based detectors

Question 11

What’s diffent in ion chromatography compare to ion exchange

Answer 11

Similar but it’s uses harsher conditions to improve the separation and sensitivity

Question 12

What is size exclusion chromatography

Answer 12

Used on large molecules where size distinguishes thing greatly

porous resin is used to separate based on hydrated radius size (not ion size)

The pore size of the resin beads determine the retention, if smaller pore size, smaller molecules penetrate and are slowed down

Larger ions pass in between the beads and are slowed down less

Question 13

In size exclusion chromatography are we separating based solely on ion size

Answer 13

No because Ions with same size but diff hydrated radius elute differently

Separating based on hydrated radius size where larger molecules elute faster

Question 14

Does size excitation chromatography use gradient elutions? (Changing ph)

Answer 14

No, it’s dependent on hydrated radius size and flow of molecules not their chemical properties

But the hydrated radius size does change depending on ph and ionic strength

Question 15

How are eluents in size exclusion chromatography chosen

Answer 15

Depending on Analyte solubility

Having high ionic strength to minimize on column interactions

Eluents with adjusted hydrated radii

Question 16

What is affinity chromatography

What needs to be changed to release the binding

Answer 16

The stationary phase has things that specifically bind to the analyte or the matrix

Ex. RNA, DNA, antibodies

To release the binding the ph and ionic strength need to be changed

Question 17

What does affinity chromatography work well for

Not work well for

Answer 17

To isolate single specific molecules or a couple similar ones (the matrix is washed out and has no retention, the target is isolated)

Not for separating a mixture of many compunds

Question 18

What are aptamers

Answer 18

Synthetic DNA/RNA that binds to other molecules

Question 19

What is capillary electrophoresis

Answer 19

Separation technique for charged compunds

Based on the mobility of ions in an electric field depending on their charge and size

Has no stationary phase and has an open tubular column

Question 20

In capillary electrophoresis no stationary phase and has an open tubular column means what

Answer 20

The A term and C term in the van Demeter equation are gone

The resolution of peaks are very high

Question 21

What are the detectors for capillary electrophoresis

Answer 21

Deep uv abosrbance

Mass spec

Question 22

What is capillary electrophoresis machine made of

Answer 22

The capillary is made of fused silica (glass) with the silica groups facing inward

It’s coated in polyimide coating which gives the glass stability

The sample is preloaded onto the capillary and flows from anode (+) to cathode (-)

Question 23

What is electrophoretic mobility in capillary electrophoresis

Answer 23

It’s the migration of cations to the cathode and anions to the anode

If something has a higher charge, higher mobility, gets to the anode or cathode faster

Negative or postive number tells the direction of attraction

Question 24

What is electrophoretic mobility dependent on

Answer 24

The voltage applied and the charge/size of the ion

Question 25

How does capillary electrophoresis work

Answer 25

The capillary had siO- instead of siOH

Meaning cations are specifically attracted to the capillary and are in the diffuse part of the capillary tube

More cations closer to the walls of the capillary causes net flow to the cathode

Question 26

What is electroosmotic flow

Answer 26

When the excess cations pump the net momentum of the analyte toward the cathode

Question 27

What would be the order of elution in capillary electrophoresis

Answer 27

The + charges ion elute first, neutral second, larger and more negative charges last

Question 28

In capillary electrophoresis, what is contributing the most to resosultion?

Answer 28

Not a or c terms so B (diffusion)

This depends on the time spent in the column which depends on flow rate

The flow rate is determined by the voltage applied

Higher voltage, higher flow rate, less diffusion

Question 29

Equations slide 18

Answer 29

Okay

Question 30

What is lab on a chip

Answer 30

Tiny channels in glass or paper let us analyze small volume samples