Harris: Quantitative Chemical Analysis, Eight Edition

CHAPTER 24:

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

How to measure Historical Ocean Temperatures

• Archaea are single-cell organisms that constitute a substantial fraction of life in the ocean. They manufacture cell membrane lipids, which can be measured by liquid chromatography.

Lipids 0 and 4 are the major components. Lipids 1 : predominant in archaea living at low temperature (near 00 C). Lipids 2,3,4’ : manufactured in increasing amounts at warmer ocean temp.

TEX 86 = [2] +[3] +[4’ ] / [1] + [2] +[3]+[4]

CHAPTER 24: Opener B

TEX 86 = [2] +[3] +[4’ ] / [1] + [2] +[3]+[4]

- Aspirin is injected into a rat. - Aspirin is converted to salicylic acid in the bloodstream. - Aspirin concentration in the blood is monitored with time.

Aspirin salicylic acid

How to measure drug metabolism

How to measure drug metabolism - Small Aspirin molecules is converted to Salicylic acid in the blood by large enzyme molecules. - Microdialysis is needed to separate the small Aspirin molecules from large enzyme molecules. It is because Aspirin would continue to be metabolized by enzymes in the blood, if you simply withdrew blood for analysis.

In Vivo Microdialysis for Measuring Drug Metabolism - A microdyalysis probe has a semipermeable membrane attached to the shaft of a hypodermic needle, which can be inserted into an animal. - Fluid is pumped through the probe from the inlet to the outlet. - Fluid exiting (dialysate) can be analyzed by liquid chromatography. - Microdialysis separates the small Aspirine molecules from large enzyme molecules.

LC-Chromatogram of blood microdialysis sample drawn 5 minutes after intravenous injection of Aspirin.

Ch. 24-0 High Performance Liquid Chromatography (HPLC)

-Solute and Mobile phase (solvent) : LIQUID - Liquid Chromatography is important because most compounds are not sufficiently volatile for Gas Chromatography. - Chromatographers generally choose gas chromatography over liquid chromatography, because the former is less expensive and generates much less wastes.

- HPLC (High Performance Liquid Chromatography) : LC using high pressure to force solvent through closed columns containing very fine particles that give high resolution separations.

Why smaller particles (stationary phase) gives better resolution ? More uniform flow through the column, thereby reducing the multiple path term (A) in the Van Deenter Equation. (Typical size: 3-5 um) → decrease plate height → increase resolution.

24-1 The Chromatographic Process

24-1. Small particles give high efficiency, but require high prerssure

The distance through which solute must diffuse in both (mobile & Stationary) phases is on the order of the particle size. The smaller the particles, the less distance solute must diffuse The smaller particles decrease the distance through which solute must diffuse in both (mobile & Stationary) phases, thereby increase mass transfer. decrease the finite equilibration term (C, C= Cs + Cm ) in the Van Deemter Equation.

24-1 The Chromatographic Process

xx

CuuBAH ++≈

xmsx uCCCuH )( transfermass +==

(a) (b) decrease the packing particle size (decrease H improve resolution) (b) (c) increase the solvent strength A stronger solvent elutes solutes more rapidly from the column (decreasing run time) *This rule applies not to GC but LC

But it needs high pressure to overcome resistance to solvent flow. HPLC requires pressures of 70-400 bar to attain flow rates of ~0.5-5 mL/min

Equation)Kozeny -(Carman )1(180 2

32

εμ

εdLΔPJ

−⋅⋅

⋅=

L : column length ∆P : pressure drop d : particle diameter µ : viscosity of solvent ε : voidage J : flux (liter/cm2⋅min)

24-1. Small particles give high efficiency, but require high prerssure

24-1. Small particles give high efficiency, but require high prerssure

Fig. Chromatograms of the same sample run on column packed with a) 10 um and b) 5 um diameter silica particles Note that how an additional peak resolved from a slow moving component.

1) Solvent delivery system (high pressure pump) 2) sample injection valve, 3) Detector, 4) Computer to control and display

24-1 The Column

Main Column : steel or plastic

: length; 5 ~ 30 cm, I.D.; 1 ~ 5 mm

: Easily degraded by irreversible

adsorption of impurities from solvent

or sample. Guard Column : collect irreversible adsorbed solutes to protect main column. Periodically replaced. (1cm long, same stationary phase as main column) Porous titanium frits : uniform flow at the junction

between narrow tubing and

wide column

- Heating a main column

lower the viscosity of solvent (µ) → reducing the required pressure (∆p) or speeding the diffusion of solutes → improve resolution → decrease retention time → degrade the stationary phase (disadvantage) → decrease column life (disadvantage)

24-1 The Column

Equation)Kozeny -(Carman )1(180 2

32

εμ

εdLΔPJ

−⋅⋅

⋅=

24-1 The Stationary Phase

Microporous particles of silica : the most common support. : permeable to solvent : surface area = several hundred m2/gram : It dissolves in base. *So it should not be used above pH=8. 1) Bare silica can be used as the stationary phase for adsorption chromatography 2) Bonded stationary phase covalently attached to silica surface for liquid-liquid partition chromatography

24-1 The Stationary Phase

a) Aggregates of spherical particles with 50 % porosity and a surface area of 150 m2 /g b) Spongelike structure with 70% porosity and a surface area of 300 m2 /g In both cases, nominal pore size (공칭공경) is 10 nm

Support:

1) Bare silica - The most common : Microporous particles of silica that are permeable to solvent and large surface area. - A silica surface has up to 8 umol of silanol groups (Si - OH) per m2.

- Exposed Si-O- groups (pH>3) strongly retain protonated bases and lead to tailing.

Bare silica

Fig. 24-7 Tailing of amine bases on silica. a) common silica support gives distorted peaks (tailing). Exposed Si-O - groups (pH>3) strongly retain protonated bases and lead to tailing. b) Less acidic silica with fewer Si-OH groups gives symmetric peaks with shorter retention time.

2) Bonded stationary phase covalently attached to silica surface for liquid-liquid partition chromatography

R = ? p. 600

2) Bonded stationary phase covalently attached to silica surface for liquid-liquid partition chromatography

R = ? p. 600

- The octadecyl (C18) stationary phase is by far the most common in HPLC. (ODS OctaDecylSilane ) - Bidentate C18 stationary phase stable in the pH range of 2-11.5

-The siloxane (Si-O-SiR) bond hydrolzes below pH 2 But, bulky isobutyl groups protect siloxane bonds from hydrolysis at low pH. Si-O-Si bond is less accessible to attack by H3O+

- Nonpolar bonded phase with embedded polar amide group provides compatibility with 100% aqueous phase. * Other nonpolar stationary phases should not be exposed to 100 % aqueous phase because they become very difficult to re-equilibrate with organic phase

Nonpolar bonded phase with embedded polar amide group

Superficially Porous Particles; - 0.25 µ thick porous silica layer + 5 µ diameter nonporous silica core - A stationary phase such as C18 is bonded to the thin, porous outer layer. - Mass transfer of solute into a 0.25 µ is 10 times faster than that into a 2.5 µ fully porous silica. - Useful for the rapid separation of proteins which diffuse more slowly.

Separation of two enantiomers

(R, R), (S,S) : Mirror image forms of the stationary phase -Binding of (s)-naproxen to (s,s) –stationary phase (left) is stronger than binding of (R)-naproxen to (s,s)–stationar phase (right). Therefore (R)-naproxen is eluted before (s)-naproxen from (s,s)–stationary phase.

- In adsorption chromatography, solvent molecules compete with solute molecules for sites on the stationary phase. - The relative abilities of different solvents to elute a given solute from the adsorbent are nearly independent of the nature of the solute. - Elution occurs when solvent displace solutes from the stationary phase (competition

between solute & solvent)

24-1 The Elution Process

1) Eluotropic series : Relative abilities of solvents to displace solutes from a given adsorbent (competition between solute & solvent) 2) Eluent Strength (ε°) : The measure of solvent adsorption energy on stationary phase. : ε° = 0 for pentane for adsorption of bare silica which is polar : With bare silica, the more polar the solvent, the greater is its eluent strength for the adsorption chromatography, the more rapidly will solutes be eluted from the column.

24-1 The Elution Process

3) Normal-Phase Chromatography : Polar Stationary phase like bare silica : Solvent is nonaqueous : A more polar solvent (increasing the polarity of nonaqueous solvent) has a higher eluent strength Reversed - Phase Chromatography : Nonpolar or weakly polar Stationary phase : It eliminates peak tailing because the stationary phase has few sites that can strongly adsorb a solute to cause tailing : Solvent is usually aqueous : A less polar solvent (decreasing the fraction of water in the mobile phase) has a higher eluent strength : Sensitive to small amounts of water in the eluent : more common

24-1 The Elution Process

* In general, eluent strength is increased by making the mobile phase more like the stationary phase.

Isocratic elution

: one solvent or a constant solvent mixture

Gradient elution

: Continuous change of solvent composition to increase eluent strength

and elute more strongly adsorbed solutes

: Increased eluent strength is required to elute more strongly retained solutes

: Analogous to temperature programming in G.C.

Ch. 24-1. Isocratic and Gradient Elution

Fig. 24-12 Isocratic HPLC separation of a mixture of 8 aromatic compounds from a reversed phase column (e.g., stationary phase; nonpolar). Solvent A; water (polar), Solvent B; acetonitrile (less polar, high eluent strength)

Decreasing the volume solvent B reducing eluent strength

Fig. 24-12 Isocratic HPLC separation. Separation becomes better. However it takes too long

Fig. 24-12 Isocratic HPLC separation. Backing up to 30 % B. It is still too long

Fig. 24-13 Gradient Elution (low high vol.% of CH3CN). 30% 45% (8-13 min), 45% 80% ( 28-30 min)

Hydrophilic Interaction Chromatography (HILIC)

* Stationary phases for HILIC are strongly polar

Hydrophilic Interaction Chromatography (HILIC)

- HILIC is most useful for small molecules that are too polar to be retained by reversed-phase columns. - The mobile phase typically contains CH3CN (25-97 vol %) or other organic solvent mixed with aqueous buffer. - HILIC is useful for separating peptides and saccharides (sugars) which are soluble in water.

24-2 Injection in HPLC

- The quality of HPLC pump: how steady and reproducible a flow it can produce. - A fluctuating flow rate creates detector noise obscuring weak signals.

24-2 Detection in HPLC Ideal detector : i) sensitive to low concentration of every analyte ii) linear response to analyte concentration iii) does not broaden the eluted peak iv) insensitive to changes in temp. and solvent composition

Linear Range : analyte concentration range over which detector response is proportional to concentration Dynamic Range : range over which detector responds in any manner (not necessarily linearly) to changes in analyte concentration

Detection Limits :the concentration that gives a signal equal to twice the peak-to-peak noise level of the baseline.

25-2 Detection in HPLC

25-2 Detectors

1) Spectrophotometric Detector (U.V. or visible detector) - Most common because many solutes absorb U.V. - linear range over which Beer’s law is obeyed is 105 - simple system: 254 nm emision of Mercury Lamp - More versile system: Deuterium, Zenon, Tungsten lamps and a monochromator

- more universal (respond to almost every solute) carbohydrates, aliphatic polymers that have little U.V absorption - detection limit is 1,000 times poorer than U.V. detector (not useful for trace analysis) - can not be used for gradient elution because base line changes as the solvent change - sensitive to changes in temp. (~0.01 0C) and pressure

25-2 Detectors: 2) Refractive Index Detector

- It responds to any analyte that is significantly less volatile than the mobile phase. -Eluate is mixed with N2 in the nebulizer and forced through a small bore needle to form uniform dispersion of droplets. -Solvent evaporates from the droplets in the heated drift tube, leaving a fine mist of solid particles to enter the detection zone at the bottom.

25-2 Detectors : 3) Evaporative Light –Scattering Detector

4) Electrochemical Detector

- It responds to reducible or oxidizable analytes (phenols, ketons, etc.)

- potential is maintained at a selected value , and current is measured.

- current is proportional to solute concentration over six orders of

magnitude

- sensitive to flow rate and temp. changes

5) Fluorescence detectors

- especially sensitive to respond only to the few analyte that fluoresce

- Derivatization : fluorescent groups can be chemically attached to

the desired analytes

*pre and post-column derivatization

25-2 Detectors

6) Inductively Coupled Plasma atomic emission

Mass Spectrometry

Fourier Transform Infrared spectroscopy (FTIR)

Flame Ionization Detector, etc

Conductivity measurement

25-2 Detectors