Introduction to Chromatographic Separations

Separation of complex mixtures Sample transported in a mobile phase gas, liquid, supercritical fluid Mobile phase / sample passes over/through an immiscible stationary phase that is fixed in place in column or on solid surface Mobile and stationary phases chosen so that sample components will distribute themselves between the two phases to varying degrees Components strongly retained by the stationary phase will travel slower & elute at longer times than those weakly retained Each component separates into a discrete band or zone that can be qualitatively or quantitatively analyzed

Chromatography Classifications
By means of contact: Column Chromatography stationary phase held in narrow tube through which mobile phase is forced under pressure (our focus) Planar Chromatography stationary phase supported on a flat plate or in a paper; mobile phase travels by capillary action or gravity By mobile/stationary phase (Table 26-1): Liquid Chromatography (LC) mobile phase is a liquid; stationary phase can be adsorbed liquid, bound organic species, solid, ionexchange resin, liquid in a polymeric support Gas Chromatography (GC) mobile phase is a gas; stationary phase can be adsorbed liquid, bound organic species, or solid Supercritical Fluid Chromatography (SFC) mobile phase is a supercritical fluid; stationary phase is bound organic species
Introduction

26A

Column Chromatography - Elution

sample A+B B A packed column B A B A t0 detector signal time t1 t2 A t3 B mobile phase addition of mobile phase washes sample through column B spends more time in stationary phase than A

B t4

detector

chromatogram
Introduction

26A

Chromatography Characteristics
Analyte dilution band broadening during separation leads to dilution of analyte; requires greater detector sensitivity
concentration

t1 A B distance migrated B

t2 A

Chromatogram plot of detector response (concentration) vs. migration time or mobile phase volume is used for qualitative (peak position) or quantitative (peak area) analysis

Introduction

26A

Migration Rates & Zone Broadening

Effect on Resolution
Improved separation changing elution time (b) or decreasing broadening (c) can each be used (a) unresolved (overlapping) peaks
detector signal

(b) Increase band separation (c) decrease band width Time

Variables that influence relative migration rates (26B) and zone broadening (26C) can be described mathematically
Introduction

26A

Migration Rates of Solutes

For a single solute A involved in transfer between mobile and stationary phases, an equilibrium is reached whose constant K is the distribution constant, partition ratio, or partition coefficient. cS Amobile Astationary K= c M Linear chromatography K is constant over a wide concentration range Retention time (tR) time for analyte to reach detector Dead time (tM) time for unretained species to reach detector Average linear rates for analyte (v) and mobile phase (u): v = L/tR Also, v=uX u = L/tM where L is length of column packing

moles of solute in mobile phase total moles of solute

Introduction

and since cV = moles

26B

Retention Time & Partition Coefficient

v=uX cMVM 1 =uX cMVM + cSVS 1 + cSVS /cMVM which is the rate of solute migration as a function of partition coefficient & volumes of stationary & mobile phases combine with definition of K to get: v=uX 1 1 + KVS /VM

Retention factor (kA) describes migration rates of a solute on a column kA = KAVS VM kA = tR - tM tM tR & tM obtained from expt.; ideally 2 < kA < 10

Selectivity factor () describes separation of two solutes on a column = KB kB (tR)B - tM = = KA kA (tR)A - tM B is always more strongly retained species, always > 1 this is correct
Introduction

26B

Zone Broadening & Column Efficiency

Kinetic (rate) theory of chromatography explains peak shapes quantitatively based on a random-walk mechanism well discuss qualitatively. Peaks are Gaussian in shape due to thousands of transfers between mobile and stationary phases; elution occurs only in mobile phase - extended residence in stationary phase longer retention times - extended residence in mobile phase shorter retention times - longer time in column broader peaks; faster flow narrower peaks

Plate theory of chromatography provides terminology: plate height H & number of theoretical plates N, related by N = L/H (L is column length) plate theory based on analogy of separation to a series of distillation plates at which solute reaches equilibrium between phases abandoned b/c it does not account for peak broadening
Introduction

26C

Column Efficiency / Plate Height

Plate Height is directly related to variance (2) or breadth of Gaussian peak (L - 1) (L + 1) 2 Analyte profile at H= L end of packing L Plate height contains ~34% of analyte (1 ) Based on time, variance is defined as 2, related to by = /(L/tR) At the base, a peaks width W = 4, and thus = LW/4tR LW2 Plate height can be defined as H = 16tR2 Number of theoretical plates N = 16(tR/W)2 Using width at half height (W1/2), N = 5.54(tR/W1/2)2 H & N used by manufacturers / researchers to measure column performance comparison can be done using same compound.
Introduction

26C

Zone Broadening / Kinetic Variables

van Deemter plot of H vs. u (linear flow rate) shows efficiency dependence on flow rate, other variables (diffusion coefficients, particle diameter, stationary phase thickness, retention factor, etc.) listed in Table 26-2 Multipath Term (A) each molecule takes a different path through column H, cm Longitudinal Diffusion Term (B/u) solute molecules diffuse to regions of Cu lower solute concentration in front of A & behind zone; inversely proportional B/u to mobile phase flow rate

u, cm/s

Mass-Transfer Terms (Cu) for Stationary Phase (CS) & Mobile Phase (CM) solute molecules require time to reach the surface of either stationary or mobile phase, depending on thickness of each phase & diffusion Zone Broadening reduced by using smaller particles, narrower columns, lower temp. in GC, thinner liquid stationary phases Introduction 26C