Harris: Quantitative Chemical Analysis, Eight Edition

CHAPTER 24:

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

CHAPTER 24: Opener A

CHAPTER 24: Opener B

How to measure drug metabolism

- Aspirin is injected into a rat- 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 metabolismSmall Aspirin molec les is con erted to Salic lic acid in the blood b large en me- 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- Microdialysis is needed to separate the small Aspirin molecules from large enzymemolecules. It is because Aspirin would continue to be metabolized by enzymes in theblood, 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.p p g p- 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 Aspirinafter intravenous injection of Aspirin.

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

-Solute and Mobile phase (solvent) : LIQUIDp ( ) Q

- Liquid Chromatography is important because most compounds are not sufficiently volatile for Gas Chromatographysufficiently volatile for Gas Chromatography.

- Chromatographers generally choose gas chromatography over liquid chromatography because the former is less expensive and generatesliquid 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.

24-1 The Chromatographic Process

Why smaller particles (stationary phase) gives better resolution ?y p ( y p ) gMore 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. Small particles give high efficiency, but require high prerssurebut require high prerssure

24-1 The Chromatographic Process

The distance through which solute must diffuse in both (mobile & Stationary)h i th d f th ti l iphases 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.

xx

CuuBAH

xmsx uCCCuH )( transfermass

(a) (b) decrease the packing particle size(decrease H improve resolution)

(b) (c) increase the solvent strengthA stronger solvent elutes solutes more rapidly from the columnp y

(decreasing run time)*This rule applies not to GC but LC

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

But it needs high pressure to overcome resistance to solvent flow.

but require high prerssure

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 lengthL : column lengthP : pressure dropd : particle diameterd : particle diameter : viscosity of solvent : voidageJ : flux (liter/cm2min)

24-1. Small particles give high efficiency, but require high prerssurebut 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 componentNote 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 : Easily degraded by irreversible

adsorption of impurities from solvent

or sampleor sample.Guard Column : collect irreversible adsorbed solutes

to protect main column. i di ll l dPeriodically replaced.

(1cm long, same stationary phase as main column)Porous titanium frits : uniform flow at the junctionPorous titanium frits : uniform flow at the junction

between narrow tubing and

wide columnwide column

24-1 The Column

Equation)Kozeny-(Carman32 εdΔPJ

- Heating a main column

Equation)Kozeny -(Carman )1(180 2 εμL

J

- Heating a main column

lower the viscosity of solvent () reducing the required pressure (p) g q p ( p)

or speeding the diffusion of solutes improve resolutionp decrease retention time degrade the stationary phase (disadvantage)g y p ( g ) decrease column life (disadvantage)

24-1 The Stationary Phase

Microporous particles of silica : the most common support.p p pp: permeable to solvent: surface area = several hundred m2/gram: It dissolves in base: It dissolves in base. *So it should not be used above pH=8.

1) Bare silica can be used as the stationary phase

f d ti h t hfor 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 /gSupport:) gg g p p p y g

b) Spongelike structure with 70% porosity and a surface area of 300 m2 /gIn both cases, nominal pore size (공칭공경) is 10 nm

1) Bare silica - The most common : Microporous particles of silica that are permeable to solvent

and large surface areaand 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(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 surfacefor liquid-liquid partition chromatography

R = ? p. 600

2) Bonded stationary phase covalently attached to silica surfacefor 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 18 y pin the pH range of 2-11.5

-The siloxane (Si-O-SiR) bond hydrolzes below pH 2

B b lk i b l il b d f h d l i l HBut, 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 groupembedded polar amide group

- Nonpolar bonded phase with embedded polar amide group provides compatibility with 100% aqueous phase.

* Other nonpolar stationary phases should not be exposed to 100 % aqueousphase because they become very difficult to re-equilibrate with organic phase

Superficially Porous Particles; - 0.25 thick porous silica layer + 5 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 is 10 times faster than that into a 2.5 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.

24-1 The Elution Process

- In adsorption chromatography, solvent molecules compete with solute moleculesfor sites on the stationary phasefor sites on the stationary phase.

- The relative abilities of different solvents to elute a given solute from the adsorbentare nearly independent of the nature of the solute.Elution occurs when solvent displace solutes from the stationary phase (competition- 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) p p gadsorbent (competition between solute & solvent)

2) Eluent Strength ()) ue S e g ( )

: The measure of solvent adsorption energy on stationary phase.: = 0 for pentane for adsorption of bare silica which is polar: 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 elutedfor the adsorption chromatography, the more rapidly will solutes be eluted from the column.

24-1 The Elution Process

3) Normal-Phase Chromatography ) g p y: Polar Stationary phase like bare silica: Solvent is nonaqueous: A more polar solvent (increasing the polarity of nonaqueous solvent): A more polar solvent (increasing the polarity of nonaqueous solvent)

has a higher eluent strength

Reversed - Phase ChromatographyReversed 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 tailingthat 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 strengthhas a higher eluent strength: Sensitive to small amounts of water in the eluent: more common

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

Ch. 24-1. Isocratic and Gradient Elution

Isocratic elution

: one solvent or a constant solvent mixture: one solvent or a constant solvent mixture

Gradient elutionGradient elution

: Continuous change of solvent composition to increase eluent strength

d l l d b d land elute more strongly adsorbed solutes

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

: Analogous to temperature programming in G.C.

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)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 longp g

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

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- A fluctuating flow rate creates detector noise obscuring weak signals.

24-2 Detection in HPLCIdeal detector : i) sensitive to low concentration of every analyte

ii) linear response to analyte concentrationiii) does not broaden the eluted peakiii) does not broaden the eluted peakiv) insensitive to changes in temp. and solvent composition

25-2 Detection in HPLC

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

Linear Range : anal te concentration range o er hich detector response isLinear Range : analyte concentration range over which detector response isproportional to concentration

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

25-2 Detectors

1) Spectrophotometric Detector (U.V. or visible detector)) p p ( )- 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

25-2 Detectors: 2) Refractive Index Detector

- 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- 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 : 3) Evaporative Light –Scattering Detector

- It responds to any analyte that is significantly less volatile than the mobile phase.p

-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 gsolid particles to enter the detection zone at the bottom.

25-2 Detectors

4) Electrochemical Detector4) Electrochemical Detector

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

t ti l i i t i d t l t d l d t i d- potential is maintained at a selected value , and current is measured.

- current is proportional to solute concentration over six orders of

i dmagnitude

- 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