Unit 3: Separations Flashcards
Distribution Constant K
[S]org/[S]aq = [(1-q)m/Vorg]/[qm/Vaq]
What is qn
The fraction in aqueous phase after n separations
qn = (Vaq/(Vorg+K*Vorg))^n
what is tm
Dead/void time.
Time for only mobile phase to pass through
What is tr
tr is the amount of time a specific substance is on a column
what is ts
Time on stationary phase.
Given by tr -tm = ts = tr’ (adjusted retention time)
What is retention factor k
= ts/tm = (tr-tm)/tm
Used to correct for variations in flow rate, column conditions, etc.
Can also be
= K (Vs/Vm)
Selectivity factor alpha
= tr’2/tr’1
Alpha must be > 1 so tr’2 much be larger than the other
How to improve resolution
- Make peaks narrower
2. Space peaks further apart
Define resultion mathematically
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
What is baseline resolution
Res > 1.5
What does plate height H describe
= varience/length column packing
Describes column efficiency
Smaller = better
What is the van deenter eq
A + B/u + u(Cs + Cm)
Define the variables in the van deenter eq
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
Importance of uniform, small particle packing
Lowers the Cm contribution in van deempter eq.
Also decreases static pools
How to improve resolution? Based on van deempter eq
- Reduce packed particle size (reduces Cm and A)
- Skinnier column (reduces A and B)
- Thinner stationary phase (reduces Cs)
- Optimize flow rate (helps u term)
Ways to vary retention factor k
Increase k2 (at expense of time) change in temp (GC) Change in MP comp. (LC)
Variations in alpha
Change SP comp
Change column T
Change MP comp
Chemical rxns (Ag+ captures)
Understanding electron capture detector
Sample eluate passes over radioactive beta particle emmitter
Ionizes carrier gas (N2 - necessary)
Selective for organics w halogens
Why choose LC?
For non-volatile or thermally unstable species (inorganics/biologicals)
Allow for more MP interaction, changing composition allows better seperation
Why is porosity important in LC?
Maximizes the area of stationary phase. Superficially porous is best
Liquid bonded phase partition chromatograph
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
Define isocratic and gradient separation
Isocratic: single MP comp. (different temps)
Gradient: changing MP composition
Can use gradient profile
Parts of HPLC
- Solvent reservoir
- Solvent degasser
- Gradient valve and solv mix
- Sample injection loop
- Guard column
- Analytical Column
- High pressure pump
- Detector
- Waste
- Data acquisition
Importance of Solvent reservoir
Ports for gas line on bottles (sparging, removes dissolved gasses)
Solvent degasses
Bubbles create dead volumes and change flow rate.
Gradient valve/mixing:
Typically increase relative amount of more strongly elution solvent (normal elution)
- Binary gradient pump
- Single pump w/ proportioning valve
High pressure pump:
Steady flow, no pulsing
Often pulse dampener
Switch Valve:
6 way connection that changes connections in order to go from a fully filled sample loop to loading it on the column
Guard column
is a short column w/ the same stationary phase between sample and analytical column to protect from contaminants
Detection methods in HPLC
UV Fluoresence (derivatization) Refractive index Electrochem Mass-spec
Extending path length in HPLC (band broadening)
Z-cell helps with that
HILIC
Hydrophilic molecules too polar for revers HPLC (for small bio molecules)
SP: high polar
MP: mixed w aq buffer
Ion exchange column
Anion exchange = anions attract
Cation exchange = attact cations
Conductivity detector
Size exclusion column
Large molecules elute faster (small molecules must go through pores)
For proteins/desalting
Affinity Column
Specific interactions bind molecules of interest
Hydrophobic interaction column
Agarose gel w polar groups in pores
Elute w/ decreasing salt concentration ( increases solubility of pore)
Developing new techniques
- 0.5 <= k <= 20
- res >= 2 (2 closest peaks
- pressure <= 15 mPa
- 0.9 <= asymp factor <= 1.5
Optimize plates, retention factor, and column/solvents
Define asymmetric factor and improvement stategies
Factor = B/A (areas split down peak)
- new mobile phase/column
- clogged frit
- guard column
- sample+MP discrepency
- Overloading
Isocratic elution
Can switch elution order with MP
Use the isocratic profile to hit the extremes of %solvent comp and temperature
Normograph
Different volume % of different solvents have same eluent strength. Shown graphically
Gradient Elution strategy
Start w scout run. Gradually increase %solvent composition over 40 min. Gradients not useful are eliminated
When to use Gradient or isocratic?
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
Segment gradient:
Hold different %solvent comp over time and elute
