Unit 3: Separations

Question 1

Distribution Constant K

Answer 1

[S]org/[S]aq = [(1-q)m/Vorg]/[qm/Vaq]

Question 2

What is qn

Answer 2

The fraction in aqueous phase after n separations

qn = (Vaq/(Vorg+K*Vorg))^n

Question 3

what is tm

Answer 3

Dead/void time.

Time for only mobile phase to pass through

Question 4

What is tr

Answer 4

tr is the amount of time a specific substance is on a column

Question 5

what is ts

Answer 5

Time on stationary phase.

Given by tr -tm = ts = tr’ (adjusted retention time)

Question 6

What is retention factor k

Answer 6

= ts/tm = (tr-tm)/tm

Used to correct for variations in flow rate, column conditions, etc.

Can also be
= K (Vs/Vm)

Question 7

Selectivity factor alpha

Answer 7

= tr’2/tr’1

Alpha must be > 1 so tr’2 much be larger than the other

Question 8

How to improve resolution

Answer 8

1. Make peaks narrower

2. Space peaks further apart

Question 9

Define resultion mathematically

Answer 9

delta tr/Wav = delta Vr/Wav
= 0.589 delta tr/W(1/2)avg

Wav represents width @ base
W1/2 avg represent width @ 1/2 peak
delta tr = seperation between peaks

Question 10

What is baseline resolution

Answer 10

Res > 1.5

Question 11

What does plate height H describe

Answer 11

= varience/length column packing

Describes column efficiency
Smaller = better

Question 12

What is the van deenter eq

Answer 12

A + B/u + u(Cs + Cm)

Question 13

Define the variables in the van deenter eq

Answer 13

A: diffusion (multple path)
B: longitude diffusion
Cs: rate of diffusion in stat. phase, lowered by thin uniformly coated SP
Cm: rate of movement through MP, lowered by uniform packing, small particles

Question 14

Importance of uniform, small particle packing

Answer 14

Lowers the Cm contribution in van deempter eq.

Also decreases static pools

Question 15

How to improve resolution? Based on van deempter eq

Answer 15

1. Reduce packed particle size (reduces Cm and A)
2. Skinnier column (reduces A and B)
3. Thinner stationary phase (reduces Cs)
4. Optimize flow rate (helps u term)

Question 16

Ways to vary retention factor k

Answer 16

Increase k2 (at expense of time)
change in temp (GC) 
Change in MP comp. (LC)

Question 17

Variations in alpha

Answer 17

Change SP comp
Change column T
Change MP comp
Chemical rxns (Ag+ captures)

Question 18

Understanding electron capture detector

Answer 18

Sample eluate passes over radioactive beta particle emmitter
Ionizes carrier gas (N2 - necessary)
Selective for organics w halogens

Question 19

Why choose LC?

Answer 19

For non-volatile or thermally unstable species (inorganics/biologicals)
Allow for more MP interaction, changing composition allows better seperation

Question 20

Why is porosity important in LC?

Answer 20

Maximizes the area of stationary phase. Superficially porous is best

Question 21

Liquid bonded phase partition chromatograph

Answer 21

Immobilizes viscous liquid (SP) by covalent attachment on solid support (particles w pores)
Covalent attachment is
Si - O - Si - R where R group is SP often alkane

Question 22

Define isocratic and gradient separation

Answer 22

Isocratic: single MP comp. (different temps)
Gradient: changing MP composition
Can use gradient profile

Question 23

Parts of HPLC

Answer 23

1. Solvent reservoir
2. Solvent degasser
3. Gradient valve and solv mix
4. Sample injection loop
5. Guard column
6. Analytical Column
7. High pressure pump
8. Detector
9. Waste
10. Data acquisition

Question 24

Importance of Solvent reservoir

Answer 24

Ports for gas line on bottles (sparging, removes dissolved gasses)

Question 25

Solvent degasses

Answer 25

Bubbles create dead volumes and change flow rate.

Question 26

Gradient valve/mixing:

Answer 26

Typically increase relative amount of more strongly elution solvent (normal elution) 1. Binary gradient pump 2. Single pump w/ proportioning valve

Question 27

High pressure pump:

Answer 27

Steady flow, no pulsing | Often pulse dampener

Question 28

Switch Valve:

Answer 28

6 way connection that changes connections in order to go from a fully filled sample loop to loading it on the column

Question 29

Guard column

Answer 29

is a short column w/ the same stationary phase between sample and analytical column to protect from contaminants

Question 30

Detection methods in HPLC

Answer 30

``` UV Fluoresence (derivatization) Refractive index Electrochem Mass-spec ```

Question 31

Extending path length in HPLC (band broadening)

Answer 31

Z-cell helps with that

Question 32

HILIC

Answer 32

Hydrophilic molecules too polar for revers HPLC (for small bio molecules) SP: high polar MP: mixed w aq buffer

Question 33

Ion exchange column

Answer 33

Anion exchange = anions attract Cation exchange = attact cations Conductivity detector

Question 34

Size exclusion column

Answer 34

Large molecules elute faster (small molecules must go through pores) For proteins/desalting

Question 35

Affinity Column

Answer 35

Specific interactions bind molecules of interest

Question 36

Hydrophobic interaction column

Answer 36

Agarose gel w polar groups in pores | Elute w/ decreasing salt concentration ( increases solubility of pore)

Question 37

Developing new techniques

Answer 37

1. 0.5 <= k <= 20 2. res >= 2 (2 closest peaks 3. pressure <= 15 mPa 4. 0.9 <= asymp factor <= 1.5 Optimize plates, retention factor, and column/solvents

Question 38

Define asymmetric factor and improvement stategies

Answer 38

Factor = B/A (areas split down peak) * new mobile phase/column * clogged frit * guard column * sample+MP discrepency * Overloading

Question 39

Isocratic elution

Answer 39

Can switch elution order with MP | Use the isocratic profile to hit the extremes of %solvent comp and temperature

Question 40

Normograph

Answer 40

Different volume % of different solvents have same eluent strength. Shown graphically

Question 41

Gradient Elution strategy

Answer 41

Start w scout run. Gradually increase %solvent composition over 40 min. Gradients not useful are eliminated

Question 42

When to use Gradient or isocratic?

Answer 42

Calculate delta t/tg where delta t = retention time between first and last peak tg = gradient time Use gradient if delta t/tg > 0.40 Isocratic if delta t/tg < 0.25 Use either if between

Question 43

Segment gradient:

Answer 43

Hold different %solvent comp over time and elute