Chapter 25 HPLC
Also a little of Ch 26
High Performance Liquid Chromatography
 GC only works on compounds that are volatile enough to enter gas phase.
 Liquid Chromatography needed for other compounds.
 Utilize packed (not open tubular) columns only.
 Smaller particles in SP = better resolution.
 Smaller particles = resistance to MP flow (slow), so high pressures are used in HPLC.
– 70 –400 bar
Column
 Steel or plastic
 Usually 5 – 30 cm long.
 Inner diameter = 1 – 5 mm
Stationary Phase
 Silica Particles
– Porous
– Spherical
 Bonded stationary phase (liq – liq chrom)
– Groups covalently attached to silica.
– Most common is C18 (octadecyl, ODS)
 nonpolar
– Other groups listed p. 612
Adsorption Chromatography
 Solvent and solute molecules compete for sites on SP.
 Solute (analyte) sticks to SP.
 When solvent (mobile phase molecule) adsorbs and displaces solute, it (solute) can be
eluted from column
 Eluent Strength:
– Measure of solvent adsorption energy.
 Adsorption chrom can be normal or reverse phase.
Normal vs. Reverse Phase
 Normal phase
– SP more polar than MP
– Less polar analytes elute fastest.
 Reverse phase
– SP less polar than MP (ie. C18 SP)
– More polar analytes elute fastest.
– Eluent strength of MP decreases as its polarity increases.
 Most HPLC uses reverse phase.
– less peak tailing
– less sensitive to water or other polar impurities.
Polarity and pH
 Remember: pH of MP will effect the charge/ polarity of many analytes due to weak acid/
base equilibrium.
 When pH = pKa, [conj acid] = [conj base]
 At pH > pKa, [conj base] > [conj acid]
 At pH < pKa, [conj acid] > [conj base]
 Depending on the analyte, acid OR base form may be charged (more polar).
 Question: At pH 7, which form of benzoic acid / benzoate, pKa 4.2 predominates?
 Which form will have a longer retention time in reverse phase chromatography?
Gradient Elution
 Change of MP composition over time of run.
 May help to elute certain analytes or give better resolution.
 Accomplished with 2 MP’s, A and B.
 Pumps mix desired volumes of A and B to obtain gradually mix over time.
 Analytes that are more strongly bound to SP require greater eluent strength.
– Less polar MP (reverse phase)
Key for the next set of Chromatograms

benzyl alcohol

phenol

3’, 4’-dimethoxyacetophenone

benzoin

ethyl benzoate

toluene

2,6-dimethoxytoluene

o-methoxybiphenyl
MP A = aqueous buffer (more polar)
MP B = acetonitrile (less polar)
Detectors in HPLC
 Spectrophotometric UV detector
– Most common detector used
– Measures absorbance of UV light as elutant flows through.
– When a UV absorbing analyte passes through, a signal is generated.
 Other types:
– Refractive Index, Light scattering, electrochemical, fluorescence, conductivity
 HPLC can also me combined with mass spec or FTIR.
Quantitative HPLC and GC
 Areas under peaks are proportional to amount of analyte.
 By calibrating the column using known amounts of analyte, the amount of analyte in an
unknown can be determined.
Quantitative Analysis with Internal Standard
 AX = area of analyte X peak
 AS = area of analyte S peak
 [X] and [S] = analyte concentrations
 F= response factor (determined by calibration)
More Types of Liquid Chromatography (Chp 26-1, 2, 3)
 Ion Exchange Chromatography
 Molecular Exclusion (gel permeation)
 Both types may be utilized on LC or HPLC.
Ion Exchange Chromatography
 Charged sites bound to SP.
 Analyte ions “stick” to these sites.
– Anion exchangers bind negative analytes.
– Cation exchangers bind positive analytes.
 Ionic strength gradient elution may be used to elute analytes from column.
– “salting off”
– Example: After your analytes are bound to the column, start a gradient in the MP from 0
– 0.50 M NaCl.
– A pH gradient may also be used.
Molecular Exclusion
 Molecules separated by size.
 SP particles have small pores.
 Smaller particles get inside the pores and take longer to elute from column.
 Larger particles can’t enter SP particles.
– Stay in the column “void volume”
– Elute faster because volume is accessible to them.
 Can be used to determine molar mass of large particles (like proteins).
– Standards run through column molar mass is recorded as a function of retention time or
volume.
Elution Equation
 V0 = void vol
 Vt = total col vol
 Vr = solute retention vol
 large molecule that does not penetrate gel, Kav = 0
 Very small molecule, Kav = 1
Homework
 Chapter 25
– Exercises: 25A
– Problems: 1a, 3, 4, 11, 12, 14
 Chapter 26
– Exercises: 26B a & c
– Problems: 3, 5, 14a, 15, 16, 18