Chromatography I

Question 1

What is chromatography?

Answer 1

Analytes distribute themselves between 2 phases

Question 2

Define stationary phase

Answer 2

Layer on supporting medium that interacts with analyte

Question 3

Define mobile phase

Answer 3

Solvent/gas that flows through supporting medium

Question 4

What is a super-critical fluid?

Answer 4

S/L/G coexists together

Question 5

What is a supporting medium?

Answer 5

Solid surface where SP is bound to

Question 6

Purpose of using chromatography

Answer 6

Check identity, purity, impurities

Question 7

2 types of analytical methods

Answer 7

Spectroscopy, Chromatography

Question 8

Briefly describe how spectroscopy works

Answer 8

Energy fired at sample, enters detector, signal processed

Question 9

Difference between HPLC and GC

Answer 9

HPLC uses liquid MP

GC uses gas MP

Question 10

Briefly describe method of chromatography

Answer 10

MP contains sample, moves through SP

Question 11

Difference between how sample moves in TLC and HPLC

Answer 11

TLC - Capillary action

HPLC - Pump moves MP

Question 12

What is the retention factor?

Answer 12

How sample distributes itself between MP/SP

Question 13

If the sample is in SP for too long…

Answer 13

RF is high so retention time is high

Question 14

Factors affecting retention

Answer 14

Composition of MP/SP
Length of column
Temperature

Question 15

Equation Kc = Cs/Cm

What does Cs and Cm stand for?

Answer 15

Cs = Conc of sample in SP
Cm = Conc of sample in MP

Question 16

Describe a chromatogram

Answer 16

Detector response to compounds eluted from column (recorded as function of time)

Question 17

How do you ensure separation?

Answer 17

Detector response needs to reach baseline between chromatographic peaks

Question 18

Describe a graph with good separation of compounds

Answer 18

Peaks are at the same RT but narrower and the detector reaches baseline before second peak is recorded

Question 19

What is separation determined by?

Answer 19

Differences in RT
Resolution (how narrow/broad peaks are)
Selectivity (differing RT)

Question 20

The equation for retention factor, k = Kc x Vs/Vm

What does Vs and Vm stand for?

Answer 20

Vs = Volume of SP
Vm = Void volume

Question 21

Another equation for retention factor, k = tR - tM / tM

What does tR and tM stand for?

Answer 21

tr = Retention time of sample
tM = Hold up/dead vol time (time MP takes from injector to detector)

Question 22

Equation: Vr = Vm + KcVs

What does KcVs stand for?

Answer 22

KcVs = Additional volume needed to elute compound

Question 23

Equation Vr = tR x F

What does F stand for?

Answer 23

F = Volumetric flow rate of MP through column

Question 24

What does the peak width express?

Answer 24

How efficiently a compound transports through column

Question 25

Describe peak widths in relation to retention times

Answer 25

Narrower the peaks, more effective the column

Question 26

Describe the chromatographic plate theory

Answer 26

An equilbrium between SP/MP analytes

Question 27

When do analytes move through column?

Answer 27

When MP, so rate which analyte moves through column = Proportional to time spent in MP

Question 28

What is a column?

Answer 28

A system with number of plates, each plate represents single partition process

Question 29

Describe relations between separation and theoretical plates

Answer 29

Efficiency of chromatographic separation inc as number of t/plates inc and height of t.plate dec

Question 30

Equation N = L/H | What does each stand for?

Answer 30

N = Number of theoretical plates per column L = Length of column in mm H - Length of column corresponding to t.plate

Question 31

Equation a = k2/k1 | What does each stand for?

Answer 31

``` a = Separation factor k2 = Retention factor of later of 2 eluting peaks k1 = RF of first eluting peak ```

Question 32

Describe 'a'

Answer 32

a = Always 1 | Larger value means better selectivity

Question 33

Equation As = w0.05/2d | What does each stand for?

Answer 33

``` As = Symmetry factor w0.05 = Peak width a 1/20th of peak height d = Distance btw perpendicular dropping from peak max/leading edge of peak at 1/20th of peak height ```

Question 34

Describe effect of As

Answer 34

As value of 1 = Symmetry

Question 35

What is peak tailing and peak fronting?

Answer 35

Peak tailing = 'As' more than 1 | Peak fronting = 'As' less than 1

Question 36

Define resolution

Answer 36

Degree of separation between 2 peaks (Rs)

Question 37

Describe effect of Rs

Answer 37

Rs = 1 means peak separation of 94%

Question 38

When does baseline separation occur

Answer 38

When Rs value more than 1.5

Question 39

Describe deficiencies of plate theory

Answer 39

Assumes rapid equilibrium between MP/SP Peak broadening ignored Effect of MP ignored

Question 40

What are the 3 causes of peak broadening?

Answer 40

Eddy diffusion Mobile phase mass transfer Longitudinal broadening

Question 41

Describe eddy diffusion

Answer 41

Causes peak/band broadening cos of multiple flow paths through full column

Question 42

Describe mobile phase mass transfer

Answer 42

Broadened peak to equilibriate too slow to from before MP moved down column, takes analyte along

Question 43

Describe longitudinal broadening

Answer 43

Narrow at intro point of column, broadened analyte zone after intro on column