Method Development in HighPerformance Liquid Chromatography

The Chromatographic Process • Diffusion in liquids is 100 times slower than diffusion in gases. Therefore, in liquid chromatography it is not feasible to use capillary columns – HPLC uses packed columns • Small particles give high efficiency but require high pressure. Typical particle sizes in HPLC are 3-10 μm

Stronger solvent than in (b)

Plate Height as a Function of Flow Rate

Number of Theoretical Plates in HPLC Under optimum conditions (near Hmin), the number of theoretical plates in a column of length L is

3500 L(cm ) N≈ d p (μm ) • Small particles reduce eddy diffusion (A term) • Small particles reduce the distance solute must diffuse in the mobile phase (C term)

Smaller Particle Size Leads to • Higher plate number • Higher pressure • Shorter run time (higher sample throughput) • Lower detection limit

Required Column Pressure The pressure required to drive the solvent through a column is

u xη L P= f π r 2 d p2 f – factor depending on particle shape and packing η – viscosity of the solvent r – column radius

The Stationary Phase in HPLC • The most common support – spherical microporous silica particles permeable to solvent. Silica dissolves above pH 8 and should not be used above this pH (special grades are stable up to pH 9 or 10) • For chromatography of basic compounds at pH 8-12, polymeric supports (polystyrene) can be used

Microporous Silica Particles

50% porosity; S = 150 m2/g 70% porosity; S = 300 m2/g Nominal pore size is 10 nm

Schematic Structure of Silica Gel

Up to 8 μmol/m2 Si-OH Protonated at pH 2-3

Uses of Silica in HPLC • Bare silica is used as the stationary phase in adsorption chromatography • In liquid-liquid partition chromatography, the stationary phase is chemically bonded to the silica surface

Bidentate C18 stationary phase stable in the pH range 2-11.5

Baseline separation of enantiomers of the drug Ritalin by HPLC with a chiral stationary phase

Bulky isobutyl groups protect siloxane bonds from hydrolysis at low pH

Superficially Porous (Pellicular) Particles • A stationary phase (e.g. C18) is bonded to the thin, porous outer layer • Mass transfer of solute is 10 times faster than into fully porous particles of the same diameter • Especially suitable for separation of macromolecules (proteins), which diffuse more slowly than small molecules

Proteins separated on C18-silica. 1 – angiotensin II; 2 – neurotensin; 3 – ribonuclease; 4- insulin; 5 – lysozyme; 6 – myoglobin; 7 – carbonic anhydrase; 8 - ovalbumin

The Elution Process • In adsorption chromatography, solvent molecules compete with solute molecules for sites on the stationary phase • Elution can be described as a displacement of solute from the stationary phase by solvent

Eluotropic Series • An eluotropic series ranks solvents by their relative abilities to displace solute from a given adsorbent • The eluent strength (ε°) is a measure of the solvent adsorption energy, with the value for pentane defined as 0 on bare silica • The more polar the solvent, the greater is its eluent strength and the more rapidly will solutes be eluted from the column

Classification of HPLC Modes • Normal-phase chromatography – Polar stationary phase – More polar solvent has higher eluent strength

• Reversed-phase chromatography – Nonpolar stationary phase – Less polar solvent has higher eluent strength

Elution Modes in HPLC • Isocratic elution – performed with a single solvent or constant solvent mixture • Gradient elution – continuous change of solvent composition to increase eluent strength (analogous to temperature programming in GC)

Example: Isocratic Separation of Aromatic Compounds by RP HPLC Solvent A – aqueous buffer Solvent B - acetonitrile

Gradient Elution of the Same Mixture of Aromatic Compounds • Same column, flow rate and solvents were used

Selecting the Separation Mode

Suppose we have a mixture of small molecules soluble in CH2Cl2

“Green” Technology: Supercritical Fluid Chromatography

Phase diagram for CO2

Capillary SFC of aromatic compounds with CO2, using density gradient elution at 140 °C

Effect of Sample Solvent • The sample should be dissolved in a solvent of lower eluent strength than the mobile phase or in the mobile phase itself

n-butylaniline

Method Development for ReversedPhase Separations • Adequate resolution of desired analytes • Short run time (high sample throughput) • Rugged (not drastically affected by small variations in conditions)

Initial Steps in Method Development 1. Determine goal 2. Select method of sample preparation 3. Choose detector

Criteria for an Adequate Separation • • • •

Capacity factor 0.5 ≤ k’ ≤ 20 Resolution Rs ≥ 2 Operating pressure P ≤ 15 MPa (150 bar) 0.9 ≤ asymmetry factor ≤ 1.5

Estimating Dead Time (Volume) Ld c2 Vm ≈ 2

Ld c2 tm ≈ 2F

F – flow rate (mL/min) dc2 – column diameter (cm)

dc = 4.6 mm

Optimization with One Organic Solvent •

Choice of organic solvent 1. Acetonitrile (low viscosity, low UV cutoff) 2. Methanol (higher viscosity and UV cutoff) 3. Tetrahydrofuran (less usable UV range, slower equilibration with stationary phase)

Optimization with Two or Three Organic Solvents • Step 1 Optimize the separation with CH3CN/buffer (chromatogram A) • Step 2 Optimize the separation with MeOH/buffer (chromatogram B) • Step 3 Optimize the separation with THF/buffer (chromatogram C)

Optimization with Two or Three Organic Solvents (cont.) • Step 4 Mix the solvents used in A, B, and C, one pair at a time, in 1:1 proportion (chromatograms D, E, and F) • Step 5 Construct a 1:1:1 mixture of the solvents for A, B, and C (chromatogram G) • Step 6 If some of the results A through G are almost good enough, select the best two solvents and mix the solvents to obtain points between those two

30% MeCN 70% buffer

40% MeOH 60% buffer

32% THF 68% buffer

1 – benzyl alcohol 2 – phenol 3 – 3’,4’-dimethoxyacetophenone 4 – m-dinitrobenzene 5 – p-dinitrobenzene 6 – o-dinitrobenzene 7 – benzoin

Nomograph showing volume percentage of solvents having the same eluent strength

Temperature as a Variable • Isocratic method development for HPLC can use solvent composition, %B, and temperature, T, as independent variables • %B and T are each varied between selected low and high values • From the appearance of chromatograms we can select intermediate conditions to improve the separation

Choosing a Stationary Phase

phenyl-silica

C18-silica

Order of Steps to Improve Separation of Two Closely Spaced Peaks 1. Change the solvent strength by varying the fraction of each solvent 2. Change the temperature 3. Change the pH (in small steps) 4. Use a different solvent 5. Use a different kind of stationary phase

Gradient Elution • Used in case of general elution problem (GEP) – mixtures of compounds with a wide range of polarities • Run a broad gradient first to decide whether to use isocratic or gradient elution • If Δt/tG > 0.25, use gradient elution • If Δt/tG < 0.25, use isocratic elution • Isocratic solvent should have composition applied to column halfway through the period Δt

Gradient Elution (cont.) Δt – the difference in the retention time between the first and last peak in the chromatogram tG – the gradient time: the time over which the solvent composition is changed

Steps in Gradient Method Development 1. Run a wide gradient (e.g., 5 to 100% B) over 40-60 min. From this run, decide whether gradient or isocratic elution is best 2. If gradient elution is chosen, eliminate portions of the gradient prior to the first peak and following the last peak. Use the same gradient time as in step 1

Steps in Gradient Method Development (cont.) 3. If the separation in step 2 is acceptable, try reducing the gradient time to reduce the run time 4. If the separation is not acceptable, it can be improved by going to a segmented gradient