CHEM 2510 / Topic 6a: Liquid Chromatography - Part 1

What are three limitations w/ simple LLE?

(1) If X in M incompatible w/ method → X must be separated from the matrix.

(2) If X > 1 in M → additional extractions to isolate each X.

(3) If complex mixture of X → separation depends on K_D → K_D of X >> for all other X.

> ff., when K_D,X ≈ K_D, other X → difficult to separate from matrix.

Why does simple LLE fail to sep X + Int even after multiple ex?

> If K_D,X ≈ K_{D, Int} → co-extraction.

> ↑ ex ↑ rec ≠ ↑ selectivity

> Volume ratio changes

∴ min purity gain → need selectivity or alt method.

How do you efficiently separate mixtures? (aka countercurrent ex)

> Extract

> Separate organic

> Add fresh initial aqueous to the organic

> Extract again

> Separate

(∴ Forces repartitioning → ↑ selectivity)

How do you not efficiently separate mixtures?

> Extract

> Separate organic

> Add fresh organic to depleted aqueous

> Extract again

> Separate

(∴ ↑ recovery only ; selectivity unchanged)

Why is called counter-current ex improves separation?

> In simple LLE, A & Int always stays in org → Int stays in org even though it doesn’t like it (despite low K_D).

> In countercurrent LLE, Int has a chance of going to aq & doesn’t mind (while A loves and stays in org) → Int goes to aq.

Differentiate mobile phase & stationary phase.

• MP: ex’ing ph that moves thru sys.

• SP: ex’ing ph that remains in a fixed pos.

> How are modern chromatographic separations achieved?

> What’s the general process of separation here?

> Cont passing MP over SP.

> Spl injected into MP → moves w/ MP → Spl components partition themselves b/w MP & SP.

Why do components A and B elute at different times in column chromatography?

> Solutes = diff interactions / properties.

> diff ret in SP.

> diff mig velocities.

> Separation over dis/time.

(Bands of A & B move thru column. A sticks less → moves faster → elutes first. B sticks more → moves slower → elutes later.)

What information does a chromatogram provide about solutes eluting from a column?

> Detector records what exits column vs time.

> Fast solute = early peak, slow solute = late peak.

> Peak area = amount, peak time = retention.

On the molecular level, why do different solutes have different retention times?

Interaction strength & frequency differ per solute.

How can analytical seps be classified?

> MP/SP physical characteristics.

> Contact method b/w phases.

> chem / phys mech driving sep.

> What physical states can MP have?

> What forms can SP take?

> Gas, liquid, supercritical fluid.

> Solid, liquid film on solid support.

How are chromatographic techniques named by physical state?

MP listed first → SP second, e.g. gas–liquid chromatography (Often just MP used, e.g. liquid chromatography, gas chromatography).

How do MP & SP contact each other in column chromatography?

> SP packed in narrow tube.

> MP pushed by gravity/pressure.

> SP = sld/liq film on solid support.

> Flow gives repeated partitioning

How do MP & SP contact each other in planar chromatography?

> SP coats flat plate (glass/metal/plastic).

> Plate in chamber w/ MP reservoir.

> MP rises by capillary action.

> Solutes move as MP climbs.

How does adsorption chromatography work?

> Solutes stick to solid SP surface.

> Stronger adsorption → slower travel.

> Weaker adsorption → faster elution.

Differentiate absorption vs. adsorption in regards to phenomenon.

> Absorption: Assimilation of molecular species throughout the bulk of the solid or liquid.

> Adsorption: Accumulation of the molecular species at the surface rather than bulk.

Differentiate absorption vs. adsorption in regards to heat exchange, reaction rate, and concentration.

> Heat exchange: Abs → endothermic ; Ads → exothermic.

> Reaction rate: Abs → occurs at uniform rate ; Ads → steadily increases & reach to eq.

> Concentration: Abs → same throughout the material ; Ads → concentration at surface of adsorbent is different from that in bulk.

How does partition chromatography work?

> Column wall coated w/ thin liq film (SP).

> MP flows thru column continuously.

> Inject spl → solutes enter flowing MP.

> Each solute repeatedly partitions b/w MP ↔ liquid SP.

> When solute spends more time in MP → it moves fwd w/ flow → move faster → elute earlier.

> When solute spends more time in MP → move flower → elute later.

How does ion-exchange chromatography work?

> Column contains solid beads w fixed charged groups = stationary phase (SP)

> SP charge is permanent.

> Mobile phase (buffer) flows thru column

> Sample is injected → ionic solutes enter MP

> Solutes w opposite charge to SP are electrostatically attracted

> Those solutes bind to SP; like-charged or neutral solutes pass thru

> Strength of binding depends on solute charge + buffer conditions

> Change salt conc or pH → counter-ions compete → bound solutes are released

> Solutes elute at different times based on interaction strength

How does size-exclusion chromatography (SEC chromatography) work?

> Column is packed w porous beads (solid, not hollow tunnels)

> Mobile phase flows around beads and into pores

> Sample enters mobile phase

> Large mols cannot enter pores → stay in flowing solvent → elute fast

> Small mols can enter pores → time spent diffusing in + out → longer residence time → later elution.

How does affinity chromatography work?

> Ligands are covalently immobilized on beads in the column.

> Sample mixture (proteins, peptides, small mols) is introduced w the MP.

> Only solutes with specific, reversible affinity for the ligand bind to the SP.

> Non-binding solutes wash through the column.

> Elution is achieved by changing conditions (competitor, pH, ionic strength).

> Target solute is released and elutes in a purified band.

How does electrophoretic separation work?

> Charged solutes are placed in a conductive medium.

> An electric field is applied across the medium.

> Solutes migrate toward the electrode of opposite charge.

> Each solute has a characteristic electrophoretic mobility.

> Differences in mobility cause solutes to separate spatially.

> Separated zones are detected as bands or peaks.

> How is a chromatographic separation monitored?

> What is a chromatogram?

Sep monitored via . . .

> Detector placed at column outlet.

> Eluting solutes enter detector.

> S recorded continuously vs. time.

Chromatogram

Plot of detector S vs t w/ each solute band as one peak.

What does each peak in a chromatogram represent?

One sepped solute band w/ peak time = ret, peak area = amount.

• What is an unretained solute?

• What is void time t_m?

• What is retention time t_r?

• What is adjusted retention time t_r^’?

• unretained solute = marker moving at MP velocity.

• t_m = t for unretained solute in MP (w/ no SP interac) to move from point of injection to detector.

• t_r = t for solute to move from point of injection to detector.

• t_r’ = t_R - t_M = additional t required to travel the length of the column beyond that required by MP.

(t_M is measured using unretained solute marker that always elutes first.)

• What is retention volume V_r?

• What is void volume V_m?

• V_r = MP vol needed to move solute from point of injection to detector

• V_m = MP vol needed to move an unretained solute to move from the point of injection to the detector.

What is baseline width w?

Width of solutes’ band measured at baseline.

Measured in t or vol depending whether t_r or V_ret is of interest.

How do you determine w?

> Draw tangents at inflection pts (steepest sides.

> Extend these tangents to baseline = intercept.

> Dis b/w intercepts = w.

How are w and w_1/2 related to σ?

w = 4σ ; w_1/2 = 2.35σ

Why is bad broadening bad?

> bb spreads solute over more t.

> Peaks get wider → wider peaks overlap more → poor res → harder to tell solutes apart.

• Why is a bigger w undesirable?

• Why is a bigger ∆t_r undesireable?

• ↑ w or w_1/2 ↑ band broadening → worse sep

• ↑ ∆t_r → worse sep

smaller w_1/2 → sharper peak ; smaller w → higher column efficiency.

What’s column efficiency?

How well a column keeps peaks narrow.