HPLC: High Performance Liquid Chromatography

Liquid Chromatography
HPLC

HPLC the mechanisms (partitioning, adsorption etc) of LC employed. HPLC (high pressure utilized).

http://www.upchurch.com/TechInfo/hplcIntro.asp#

Degassing System

Spectrophotometric Detectors: UV

Variable/fixed wavelength

Instrument Components

HPLC uses packed columns predominantly dp = 1-5um L = 5 30 cm i.d = ~0.5 cm Higher temperatures (~10oC) lowers viscosity, lowers pressure used, lowers retention times, may increase bleeding, needs preheating of mobile phase.

http://www.chemistry.adelaide.edu.au/external/soc-rel/content/hplc.htm

The classification of chromatographic modes according to the retention mechanism... Liquid Chromatography
Adsorption Partition Size Exclusion Affinity
Molecular Lock and Key Equilibration Equilibration sieving between liquid between liquid mechanism based on the mobile phase mobile phase and liquid size of analyte and solid adsorbent stationary phase of the solute of the solute

Ion Exchange
Equilibration between liquid and ionic stationary phase To the solute

Stationary phase coated or bonded to the um size silica/polymer particles (base inert particles).

Capacity Factor
kR = (tR - t0) / t0 = (VR - V0) / V0

Van Deemter Equation: (Packed Column)

H column = A + Bu 1 + Cu

Selectivity (Relative retention)

= (t2 - t0) / (t1 - t0) = (V2 - V0) / (V1 - V0) = k2 / k1

Efficiency
N = number of theoretical plates; H= L / N

H col = ( d p ) +

( 2D M ) +
u

Resolution
R = 0.25*((-1)/)(N1/2)(k/(1+k))

k'2 d p qk' d f2 u + 2 (1+ k') D 96D (1+ k') 2 S M

H col = d p +

2D M qk' d u+ u + u (1+ k') 2 DS 96D M (1+ k') 2

2 f

k'2 d p

H col = ( d p ) +

( 2D M ) +
u

k'2 d p qk' d f2 + u (1+ k') 2 D 96D (1+ k') 2 S M

Diffusion coefficient Dm is low in LC. Equilibration slow; m.p. being a liquid use of capillary column is not possible (pressure considerations). Large s.p. surface needed, coated on inert particles, both df, dp non-zero. Small particles (to increase s.p. surface and minimize multiple paths and hence minimize term A) requires very high pressures to move the m.p. through the column. Lower df is preferred; faster equilibration.

Small dp, low A Small dp df; low C-slope. Uniform flow (A). Fast equilibrations because of the smaller distances analyte molecules would travel due to better packing of smaller particles (C). Larger optimum flow rate.

However these systems require a high pressure pumps to push the mobile phase through the column.

Stationary Phase Column support: small, pure, spherical, microporous particles of silica or polymer particles. Often these particles are coated with s.p. or s.p is chemically bonded to the surface. Stationary phase coating on silica particles not durable. Large surface area per mass unit. Sometimes silica particles used as is for (adsorption mechanism, and normal mode) LC.

P= f

u L 2 r 2 d p

Adsorption chromatography, NP
Si OH

Highly polar

pH > 8, silica dissolves polymeric supports pH 2-3, Si-OH pH>3 de-protonation occur may result in tailing of positively charged species pH 2-8 optimum range

Partition chromatography on bonded phases:

Si OH

Me

Me

+ Cl Si R
Me

Si O Si R Me

Chemically bonding the hydroxy groups would generate a rugged s.p. and most importantly the thinnest possible coating, df. Long carbon chains form the basic s.p. material C4 C18. Derivatization of the terminal carbons allows us to tailor s.p.s with different polarities. Such derivatized s.p. are used in the reverse phase mode, generally.

Hydrolyses pH <2

Me Si O Si Me
s.p. Monolayer!

R = long C chain with/without functional groups.

Partition chromatography, RP

R=C18

Better protected vs hydrolysis

Some common R groups (CH2)17CH3 (CH2)7CH3 (CH2)3C6H5 octadecyl octyl phenyl nonpolar RP

Well protected vs hydrolysis

(CH2)3NH2 (CH2)3CN

amino cyano diol

polar

NP

(CH2)2OCH2CH(OH)CH2OH

End-capping The procedure employed following the primary bonding process of the stationary phase onto the base inert packing material in a column.
Bonded particles

The purpose is to remove residual silanol groups from the packing surface. This process typically helps increase a column's overall CARBON LOAD.

Mobile Phase: Unlike in GC, the m.p. in LC influences the separation by actively participating in the elution of analytes from the s.p. Both s.p. and m.p. must be considered in determining the separation process. Modes of LC; normal phase - NP (s.p. > m.p. polarity) reversed phase - RP (s.p. < m.p. polarity)

Eluent Strength of solvent (m.p.)

Adsorption LC / Normal phase LC

The ability to draw analytes to the mobile phase (solvent) depends on the polarity, pH etc. Elutropic series - a relative ranking of HPLC solvents ranging from non-polar to very polar. Property based on silica s.p.. Polarity effects are due, in part, to dielectric constant, dipole moment and Hydrophobic hydrophillic properties.

In RP the strength of the eluting power of the solvent is in reverse order; less polar solvent elutes stronger.

Isocratic Elution: use of a constant-composition mobile phase in liquid chromatography. Gradient Elution: technique where mobile phase composition over time during the chromatographic separation is changed. Also known as solvent programming. Gradients can be continuous or stepwise. Binary, ternary, and quaternary solvent gradients have been used routinely in HPLC.

Isocratic elution:
Eg: Aromatic compounds

The mode is isocratic if only one composition solvent mixture or pure solvent is used! Various solvents can be used based on the best polarity ratio of the solvent mixture in the separation.

Solvent: A+B

A = aqueous buffer; B = acetonitrile

Based on the observations of the above isocratic runs, a gradient program for decreasing separation time by changing mobile phase composition over time during the chromatographic separation can be;

Load and Inject positioning

Flow cell:

Iin

Iout,solvent Iout,solution

More versatile, full spectrum

Refractive index detector: isocratic only

Evaporative light scattering detector:

uniform Response ~ mass nonlinear curves volatile buffers/solvents only no solvent peak

Fine mist of particles

Measures the scattered light

Derivatizing of samples has become vitally important in the pharmaceutical, chemical and agricultural industries. Derivatizing of reactive analytes for HPLC shows following advantages: increase in analyte stability Leads to higher selectivity and sensitivity caused by reduced polarity that leads to better chromatographic separation and/or detection. HV

Drying gas

To MS
1 torr 10-3 torr 10-5 torr

Pump

Pump

Pump

Monolithic Columns: Monolithic columns consist of a single, rigid or semi-rigid, porous rod. Monolithic columns approach fast analysis by bypassing the limitations imposed by pressure via through-pores, which allow higher flow rates than particulate columns at reasonable column backpressures. Two types of monolithic columns have been developed for chromatography: organic polymers based on polymethacylates, polystyrenes or polyacrylamides, and inorganic polymers based on silicates. SEM Micrographs of a Monolithic Stationary Phase

Made by polymerizing and precipitating the stationary phase within the separation capillary. Stationary phase is a continuous interconnected skeleton with large through-pores. the resulting flow channels have both a higher permeability and a lower impedance to bulk flow (lower back pressure), than the stacked spheres of particulate materials. This structure reduces the diffusion path. The integral structure enhances the mechanical strength.