Extraction Techniques Liq-liq extraction (LLE)Remove compound from undesired materials Solvent must have: 1. Low solubility (miscibility) in water 2. Moderate volatility, high stability and low reactivity 3. Low flammability and toxicity 4. Inexpensive and available in required purity
Rxn Quotient,Q and Equil. Constant, K
Dist. Ratio and Dist. Constant ,
,K is the same but at equilibrium. K>Q, Q/K<1 form more product, K1 form more reactant, K=Q, Q/K=1, equilibrium
Introduction to Chromatography Migration rate Void time (tM): Time for solute to move through the column Stationary phase time (tS): Time the compound spends in the stationary phase Retention time: Time between injection and appearance of component peak in detector. Refer to eqn. 1 Refer to eqn. 2 and 3, ̅ and u is the migration rate of solute and mobile phase respectively, L is column length Volumetric Flow Rate, F (cm3min-1) Refer to eqn. 4, 5, 6 , u0 is the linear flow velocity (cm min-1) Refer to eqn 7, u is the migration rate of mobile phase Retention Factor, k – Affinity the solute has for the stationary phase. Refer to eqn 8
Buffer Eqn pH=pKa+lg Acid Extraction or base Refer to eqn 6 and 7, αHa =
Conc. in left in aq phase, [A]i Refer to eqn 1 and 2 Fraction extracted, FA Refer to eqn 3 and 4 Extraction eff.=FAx100%
Selectivity factor, α- How well the column separates 2 solutes Refer to equation 9, note that α>1 since B is more strongly retained than A Plate Theory - At each plate, equilibrations of the component between the stationary and mobile phase occurs - Column length and flow rates are fixed - An assumption because there’s not time for equilibration in the column - Separation improves as N increases, L decreases and H decreases Refer to eqn 10, 11, 12, 13, H, N is the height and number of the plates respectively
Gas Chromatography (GC) Detectors Flame ionization (FID)- Highly sensitive but cannot detect non-C compounds Electron Capture-Lowest detection limit Thermal conductivity (TCD) Non destructive of sample but has low sensitivity Mass Spec-Destructive of product
High Performance Liquid Chromatography (HPLC) Column Chromatography- Stationary phase held by a narrow tube and mobile phase is forced through the tube under high pressure or by gravity. Characteristics of HPLC Sample is dissolved in mobile phase, mobile phase is an organic or aqueous solvent, stationary phase is a liquid on solid or solid beads in column, runs at several hundreds atm to pump liquid through the solid particles Organic
Fraction remaining, GA Refer to eqn 5, i=no. of extractions
Aqueous
Nonpolar
Moderately polar to nonpolar
Non-ionic or ion-paired
Ionic
Revers ed phase
CH3Cl
-OH, CH3CN, C2H5OCOCH3
Adsor ption
Normal phase
Normal phase or hydrophilic interaction
Ion exchan ge
Types of HPLC 1. Solid-liquid (adsorption)- Solutes adsorb to support surface 2. Liquid-liquid (partition)- Solutes partition into a non-polar or polar coating - Reversed-phase- Non-polar stationary phase, polar solvent - Normal- Polar stationary phase, non-polar solvent 3. Ion exchange- Charged solutes bind to fixed charges
Acid Extraction or base Refer to eqn 6 and 7, αHa =
is the
fraction of HAin aq phase
is the fraction of
HAin aq phase Metal ion extraction eqn 8 pH1/2-pH at which ½ metal ions are extracted in org. layer. Use of dithizonate in CCl4
Column Efficiency, H (lower, more efficient) and Theoretical Plate No., N - Chromatographic peaks are Gaussian so efficiency can be seen as the breadth of peaks (variance per unit length) - Peak shows the distribution of molecules at the instant the analyte reaches the detector. Refer to eqn 11, 12, 13, W is the width, W1/2 is the width at ½ height - Good separation depends on large N, good separation when peaks are sharp Deviations: Fronting and tailing Factors: - Random motion of components as they move down - Residence time at each plate is irregular - Overloaded column Deviations result in poor separation and less reproducible tR
Kovat’s Retention index Unk is unknown, N and n the no. of C in larger and smaller alkane, tR’=tS Refer to eqn 1
4. Size exclusion- Porous support separates solutes based on size 5.Affinity- Solute selectively bind to a biologically-related ligand Packing types Porous- mobile phase flows around particle Perfusion- large and small pores in larger particles. Flow around and through, high surface area Non-porous- silica or resin with small particle size. Faster flow since small s.a. compared to porous ones. Back pressure can occur Monolithic Column- continuous columns of porous silica instead of particles. Fast flow and low back pressure Silica SiOH groups are protonated at pH 2-3 so when pH>3, positively charged analyte have higher tR. Bare silica is used for adsorption Liquid-liquid (partition) Liquid can be adsorbed or chemically bonded to the silica (liquid-bonded phase partition) to increase the lifespan and reduce column bleed
Allows for selectivity of metal ion Best when pH1/2 differs by 3 or more units. If not, masking agent, selective to the interfering ion is used to maintain selectivity and quantitative separation of desired analyte. Add another chelating agent that binds to unwanted ions, forming a more stable complex that’s charged and soluble in aq.
Masking eqn 9 Where 1=before and 2=after addition of agent LLE Emulsions form due to low interfacial tension, invert flask instead of shaking, use continuous extractor Ion Exchange Resins Cation exchange uses resins with acid groups (strong: sulfonic acid; weak: carboxylic acid) Anion exchange uses resins with base groups (strong: quarternary amine; weak: secondary or tertiary amine)
Band Broadening - H increases since σ2 increases, refer to eqn. 11 - H is affected by u, diffusion coeff. in mobile and stationary, DM and DS (cm2s-1), k, diameter of particle and coating on stationary phase dp, df Linear flow rate, u Minimum H occurs at faster flow rate for gas but H is larger for gas-> GC column is longer than HPLC Rate Theory: Van Deemter Eqn -Rate theory provides info on the effect of flow rate, u on H For high flow rate, refer to eqn 14 For low flow rate, refer to eqn 15, , Cs and Cm is the mass transfer coeff. of the stationary and mobile phase (s-1), A is eddy diffusion coeff., (cm) B is the longitudinal diffusion coeff.(cm2s-1), u is linear velocity (cms-1) -Lower H, higher column eff., less band broadening Eddy diffusion, A Refer to eqn. 16, λ=consistency of packing, dP is the diameter of particle *A=0 for open tubular columns Advantages Efficient, fast, can be automated Selective, widely applicable Requires small sample size Non destructive (TCD) detectors
Mn+ (aq) ⇌ H+ (aq) + Mn+ (resin) Anionic exchange: An- (resin) + n OH- (aq) ⇌ An- (aq) + OH- (resin) KD increases with increasing ionic charge and decreasing hydrated ion radius.
Longitudinal diffusion, B Refer to eqn. 17, γ=consistency of packing, DM is the diffusion coeff. in mobile phase (cm) Mass Transfer, C Term comes from the finite time required for equilibration between both phases Refer to eqn 18, d=diameter, df(replace d when liquid)=thickness of film Solving for A, B and C Simultaneous equation with 3 points on the plot Assuming an open column, eqn 5 Refer to eqn. 13, 14 and 10 Peak Resolution, RS-Degree of separation between 2 adjacent peaks Quantitative measure of the ability to separate components Refer to eqn. 20, 21, 22, where B is slower than A RS is affected by N, kB, and α Refer to eqn 9, 10, 8 Improving Separation - Decrease peak width, increase N, use optimal flow rate, van deemter - Increase peak separation, increase kB and α, change composition of mobile phase, temp, compostion of stationary phase, special chemical effects
Disadvantages Temp, T Sample must be volatile Lower, high RS but Sample must be thermally stablelong tR Fair for qualitative analysis Length, L Diff. for large samples
e.g. of bonded groups- cap with trimethylsilyl (non-polar), bidentate C18 (nonpolar, high pH stable), Isobutyl (block H+ from attacking Si- O bond so, low pH stable, non-polar) chromatography Eluent Strength How easily a solvent displaces a solute -> fills up space in stationary phase, similar polarity as stationary phase ->lower tR, lower resolution Elution Isocratic (Constant solvent composition)- Opposite polarity from the stationary phase, lower eluent strength high resolution, low tR. Takes way too long to elute! Solution: Gradient-> Increase the eluent strength of the solvent continuously or in a series of steps. Elution order and solvent composition prediction Carry out TLC, trial and error Instrument Parameters 1. Column type- Packing type and pore size in nm- porous, perfusion, non-porous and monolithic 2. Diameters of packing particles (1.5-12 μm) 3. Coating- polar or non-polar stationary phase 4. Inner diameter (1-5mm, commonly 2.1mm) 5. Length (5-30cm) 6. Temp.- Isothermal (35 to 60oC) or programmed Heating reduces viscosity of solvent, faster flow rate and shorter tR Not too high to prevent degradation of column Mobile phase is heated before entering column to allow for consistent tR 7. Solvent- Isocratic or gradient Linear flow rate (cms-1)
Equilibrium involving In Exchange Cationic exchange: n H+ (resin) +
Longer, high RS but long tR
Column Thin film narrow borehigh RS but low capacity Thick film narrow/wide bore-good/high capacity but moderate/low tR
GC Flame Ionisation Detector Analytes are pyrolysed and form cations and electrons. Collector electrode will capture the charge carriers and the resulting current is measured. The response is proportional to the no. of carbon atoms. Thermal Conductivity Detector Carrier gas has a much higher thermal conductivity than the analytes. Thermal conductivity is lowered with the presence of organic compounds, temperature will increase and electrical resistance of the heated element increases Electron Capture Detector β-Emitter (usually Ni-63) emits electrons. Carrier gases ionise and form cations and more electrons. Analytes ionize and from anions and less electrons. The response is the frequency f the voltage pulses varied to maintain constant current
Elution in GC A polar liquid stationary phase elutes analytes with increasing polarity. A non-polar liquid stationary phase elutes analytes with increasing volatility. Effect of each Instrument Parameter Column Type: Packed column has A; capillary column no A. H is generally higher for packed column. Inner Diameter: Increasing d ↓es Ds, ↑es Cs, ↑es H, ↓es N, ↓es Rs, ↑es tR. Thickness of Stationary Phase: Increasing dp / ds ↓es Ds, ↑es Cs, ↑es H, ↓es N, ↓es Rs, ↑es tR. Length: Increasing L ↑es N, ↑es Rs, ↑es tR. Temperature: Increasing T ↓es Rs, ↓es tR. Types of Capillary (Open Tubular) Columns in GC FSOT (Fused Silica): No coating WCOT (Wall Coated): Coated with stationary liquid phase SCOT (Support Coated): Coated with stationary liquid phase on solid support PLOT (Porous Layer): Coated with stationary solid phase
