lecture 12: analytical seperations

Question 1

what is chromatography?

Answer 1

principle of extraction by keeping one phase held in place and moving the other

Question 2

What is the mobile phase?

Answer 2

(diluent) is a solvent (either gas or liquid) flowing through the column

Question 3

What is stationary phase?

Answer 3

stays in place in the column

Question 4

What is eluent?

Answer 4

the fresh solvent put into the chromatography

Question 5

What is eluate?

Answer 5

the solvent flowing out of the chromatography

Question 6

How does chromatography separate solutes?

Answer 6

one solute will be less strongly retained by the stationary particles, making it emerge first
the other solute will be retained by the stationary particle, emerging later

Question 7

What is elution?

Answer 7

the process of passing liquid or gas through a chromatography column

Question 8

what is a packed column?

Answer 8

it is filled with particles of stationary phase

Question 9

What is an open tubular column ?

Answer 9

a narrow, hollow capillary with stationary phase coated on the inside walls

Question 10

What is adsorption?

Answer 10

looking at how much it likes to sit on the surface of stationary phase

Question 11

What is adsorption chromatography?

Answer 11

stationary phase: solid (silica)
mobile phase: liquid or gas
checks the adhesion of a molecule to a surface, the higher the adsorptivity, the more time it spends in the column

Question 12

What is partition chromatography?

Answer 12

stationary phase: liquid polmer bonded on solid support
mobile phase: liquid or gas
analyte partitions between stationary liquid and the mobile phase, the boiling point, vapor pressure governs its retentions in the SP

Question 13

What is ion-exchange chromatography?

Answer 13

stationary phase: solid linked with anions and cations
mobile phase: liquid
the charge of the resin ions is balanced, and will separates analytes. Use pH or salt to release the proteins

Question 14

What is size exclusion chromatography?

Answer 14

stationary phase: porous gel
mobile phase: liquid or gas
seperates the molecules by size, the larger molecules will pass quickly.

Question 15

What is affinity chromatography?

Answer 15

stationary phase: covalently bound molecules that use molecular recognition
mobile phase: liquid
examples: antibodies, (used in protein purification)

Question 16

What is retention time?

Answer 16

amount of time a specific component spends in the column

Question 17

What are the two factors that contribute to how well compounds are seperated by chromatography?

Answer 17

1. diffeence in elution times between peaks (further apart the better)
2. how broad the peaks are: the wider the peaks, the poorer the separation

Question 18

What are theoretical plates?

Answer 18

imaginary one in which a solute reaches equilibirum between the mobile phase and the stationary phase. The more plates the more effective

Question 19

How do you calculate the number of theoretical plates?

Answer 19

N= 5.55T2/w2

Question 20

How are theoretical plates calculated?

Answer 20

H=L/N whre H is the plate height, N is the plate number and L is the length of column

Question 21

what is resolution?

Answer 21

measure of how clearly two peaks are separated. The higher the resolution the better

Question 22

What is a classic LC?

Answer 22

open columns, make a slurry, dont let the top of the column dry

Question 23

What is a HPLC?

Answer 23

high pressure closed column, using pressure to force the solvent through the closed column. This gives high-resolution separation

Question 24

What quantitative information can you get from LC?

Answer 24

calibration curve (used to purify)

Question 25

What qualitative information can you get from LC?

Answer 25

analyte identification, sample classification, size, relative chemistry

Question 26

What does a HPLC consist of?

Answer 26

- a solvent delivery system (pump) - a sample injection valve - a high pressure chromatography column - a detector - a data system

Question 27

What are injection valves?

Answer 27

they introduce the samples into the column

Question 28

What is the stationary phase of HPLC?

Answer 28

micropourous silica particles, they are pure and porous allowing the solutes to enter

Question 29

What is the most common form of HPLC?

Answer 29

liquid-liquid partition chromatography

Question 30

How does size of particles correlate to resolution?

Answer 30

the smaller the particles, the better the resolution, the tradeoff, you need more pressure

Question 31

In HPLC what is the normal phase chromatography?

Answer 31

using a polar stationary phase and a less polar solvent, it will have the ability to push the analyt out of the column by replacing it

Question 32

In HPLC what is reverse phase chromatography?

Answer 32

using a non-polar stationary phase and the solvent is polar, the mobile phase is stronger.

Question 33

What is isocratic elution?

Answer 33

using a single or constant solvent composition to use on simple samples that are already well-separated using a single mobile phase composition

Question 34

What is a gradient elution?

Answer 34

using a continuous change of solvent composition to increase the mobile phase eluent strength, used to seperate mixtures that have a wide range of polarities

Question 35

What is gas chromatography?

Answer 35

separating and analyzing volatile compounds in a mixture. Mobile phase: gas, stationary phase: solid or liquid. The analytes must have a boiling point of less than 400ºC

Question 36

What does the column inner diameter and resolution have in common?

Answer 36

if the column is too thick, stationary phase and solvent wont interact. the narrower and longer the column the better the resolution.

Question 37

What is the correlation between stationary phase thickness and resolution?

Answer 37

thicker stationary phase provide better resolution for early-eluting peaks thinner stationary phase provide better resolution for later-eluting peaks

Question 38

How do you choose a stationary phase?

Answer 38

like dissolves like nonpolar solutes - nonpolar SP intermediate - intermediate SP etc

Question 39

What do column bleeds cause?

Answer 39

background signal, reduces the signal to noise ratio, and gradually contaminates the detector.

Question 40

What are the three common types of injections?

Answer 40

1. split injections: a portion of the injected sample is discarded 2. splitless injections 3. on-column injections: used for quantitative analysis

Question 41

What is solvent trapping?

Answer 41

you slowly raise the temperature of the column, with the solvent inside, match the solvent polarity to the column polarity

Question 42

What is cold trapping?

Answer 42

you rapidly increase the temperature, solvent and low boiling components are eluted rapidly and highboiling solutes remain in the column

Question 43

What are the common gas chromatography detectors?

Answer 43

1. thermal conductivity: measures the change in voltage 2. Flame ionization: producing ions and hydrogen flame; commonly used for detecting hydrocarbons and volatile organic compounds (destroys sample) 3. Electron Capture: looking at frequency of pulse between anode and cathode, sensitive to halogen containing molecules 4. mass spec