High Performance Liquid Chromatography
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Transcript of High Performance Liquid Chromatography
High performance liquid chromatography
Daniel Zahn
Idstein | Köln | Hamburg | Düsseldorf | München | Frankfurt am Main | Berlin | Zwickau | New York
Agenda
Principles of Chromatography
Terms and definitions
HPLC techniques
Instrumentation
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Practical applications
Idstein | Köln | Hamburg | Düsseldorf | München | Frankfurt am Main | Berlin | Zwickau | New York
Origin of chromatographic separation techniques
Mikhail Tswett (early 19th century):
• Investigation of plant extracts with calcium carbonate filled columns and petrol ether/ethanol
• Zones with different colors were observed
• Further investigation and application as separation technique
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• Technique was termed chromatography, derived from the greek word chroma (color) and graphein (to write)
Principle of separation
• A chromatographic system consists of a mobile phase and a stationary phase
• Chromatographic separation is based on the interaction of sample molecules with the stationary phase
• The mobile phase can interact with the sample molecules as well or influence their interactions with the stationary phase
• sample molecules travel with the mobile phase but are retained by the stationary phase
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• Retention by the stationary phase can in most cases be attributed to at least one of the basic mechanisms:
• Adsorption• Distribution
Adsorption
Distribution
Adsorption chromatography
• Stationary phase has active centers of any kind that allow adsorption of sample molecules
• Sample molecules adsorb to and desorb from the stationary phase repeatedly
• Properties of the sample molecules (affinity to stationary phase) determine the frequency of adsorption
• Molecules with a higher affinity to the stationary phase are adsorbed more frequently and therefore more strongly retained
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frequently and therefore more strongly retained
• Molecules with a low affinity to the stationary phase spend little time adsorbed and thus are quickly transported by the mobile phase
Distribution chromatography
• Stationary phase is a liquid attached to a solid support
• The stationary and the mobile phase have to be immiscible
• Continuous distribution of sample molecules between the mobile phase and the stationary phase takes place
• Molecules with an distribution equilibrium shifted towards the mobile phase spend the majority of the time in the mobile phase and are therefore transported rapidly
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• Molecules with an distribution equilibrium shifted towards the stationary phase spend the majority of the time in the stationary phase and are therefore strongly retained
Classification of chromatographic techniques
Classification by mobile phase state of matter:• gaseous (Gas chromatography, GC)• liquid (Liquid chromatography, LC)• supercritical fluid (Supercritical fluid chromatography, SFC)
Classification by phase pair (mobile phase/stationary phase):• Liquid/solid (Liquid-solid-chromatography, LSC)• Liquid/liquid (Liquid-liquid-chromatography, LLC)• Gas/solid (Gas-solid-chromatography, GSC)• Gas/liquid (Gas-liquid-chromatography. GLC)
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• Gas/liquid (Gas-liquid-chromatography. GLC)
Classification by application technique:• Planar chromatography (e.g. thin layer chromatography, TLC)• Column chromatography (e.g. HPLC, GC)
High Performance Liquid Chromatography (HPLC) is column chromatography with a liquid mobile phase and solid stationary phase or
liquid stationary phase attached to a solid support
The chromatogram
Inte
nsity
t0 dead timetR retention timet`R reduced retention timeA peak areah peak heighth1/2 half peak heightw peak widthw peak width at half height
A chromatogram is a plot of the intensity against the time. It can be described by a variety of basic parameters:
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time
w1/2 peak width at half heightk` capacity factor
The capacity factor is often more suitable than the reduced retention time to describe
the retention behavior of a substance because it is independent of the column length and flow rate. The capacity factor
should be kept between 1 (better 2) and 10 during method development
Peak types
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Fronting and tailing
Fronting (AS < 1): Overload of mobile phase, instrumental problems
Tailing (AS > 1): Overload of stationary phase, secondary interactions, instrumental problems
Reasons for fronting and tailing are diverse and have to be investigated separately for any case. Tailing is much more common than fronting.
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Fronting and tailing may be expressed as asymmetry factor (IUPAC) or tailing factor (USP). Both calculations are valid but a specific calculation may be requested by regulatory authorities.
Strong fronting and tailing has adverse effects on peak high and resolution and thus severely compromises quantification.
Number and height of theoretical plates
• Chromatography is based on continuous interactions of the analytes with mobile and stationary phase
•Theory of plates is deployed to simplify the description of the continuous process
• The chromatographic column is viewed as consecutive sections in which an equilibrium is reached. The height of these sections is the height of a theoretical plate (H) and their quantity is the plate number (N)
H
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number (N)
l column length
The plate number significantly influences
the broadness of a peak
Van-Deemter-equation
height of a theoretical
plate H
High plate numbers result in narrower peaks. The plate number can be optimized by increasing the column length or decreasing the plate height.
Van-Deemter-eqiation:
H = A + B/u + C*u
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mobile phase flowrate u
optimal flowrate
minimal plate height
A eddy diffusion
B longitudinal diffusion
C mass transfer
Van-Deemter-equation
Eddy diffusion:Different path lengths due to variety of possible ways through the columnIndependent on flow rate
Longitudinal diffusion:Diffusion of sample molecules in or against the mobile phase flow rate
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phase flow rateEffect is significantly increased at low flow rates
Mass transfer:Mass transfer in and out of stationary phase/stagnating mobile phase in stationary phase poresEffect is increased at high flow rates
Van-Deemter-equation
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Chromatographic resolution
• The resolution describes the separation of two adjacted peaks
• A minimal resolution of 1.5 is required for baseline separation of Gaussian peaks
• Baseline separation is a requirement for accurate area and height measurement
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Baseline separation is mandatory if quantification
is performed with non-selective detectors!
Optimization of resolution
The three important factors for the resolution are:
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resolution are:
• the efficiency
• the retention factors
• and the selectivity
Optimization of efficiency
A high efficiency results in narrow peaks and therefore a better resolution.The efficiency can be improved by:
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• increasing the column length (also increases analysis time!)
• deploying smaller particles (also increases backpressure!)
• optimization of the flow rate
• other factors
Optimization of the retention factor
A very low retention factor is a consequence of (almost) no interactions with the stationary phase and therefore no separation. Once retention is sufficient (k > 5) a further increase has almost no influence on the resolution
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almost no influence on the resolution
The retention factor can be improved by:
• adjusting the solvent strength (effective and simple!)
• changing the stationary phase
Optimization of the selectivity
The selectivity is the ability of a chromatographic system to distinguish between two substances. It is the most efficient but most complex way to improve the resolutionThe selectivity may be improved by:
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• changing the organic solvent
• changing the pH value
• changing the stationary phase
• changing the column temperature
• other factors
Every influence on the selectivity is substance specific, therefore any of the listed changes may or may not influence the selectivity of a
specific substance pair.
Reversed Phase HPLC
OH2
OH2
OH2
OH2
OH2
OHOH2
OH2
OH2
OH2
NCH3
NCH3NCH3
NCH3
NCH3
NCH3
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OH2
NCH3NCH3NCH3
Stationary phase: non-polar (e.g. C18, C8)Mobile phase: polar to semi polar (mixtures of H2O, ACN, MeOH, additives)Analyte: Substances of intermediate to low polarityMechanism: • Non-polar stationary phase acts as immobilized liquid• Liquid-liquid-partition between stationary and mobile phase takes place• Secondary interactions (e.g. with underivatized silanol) are possible
Normal Phase-HPLC
CH3 O
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
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Stationary phase: polar (e.g. SiO2,Al2O3)Mobile phase: non-polar (e.g. hexane)Analyte: polar (has to be soluble in mobile phase)Mechanism:• Polar molecules tend to attach to one another in a non-polar environment• The polar analytes adsorb to the polar surface of the stationary phase
CH3
CH3
CH3
Ion chromatography
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Stationary phase: charged polymersMobile phase: aquatic buffer solutions (e.g. NaHCO3)Analyte: ionicMechanism:• Surface of the stationary phase is charged• Analytes of the opposite charge are attracted to the stationary phase
Hydrophilic interaction liquid chromatograph
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Stationary phase: polar (e.g. SiO2, polar modifications)Mobile phase: polar to semi polar (mixtures of H2O/ACN/MeOH)Analyte: polarMechanism:• Water is strongly retained on the surface of the stationary phase• partition equilibrium between the mobile phase and the immobilized water layer on the stationary phase
Gel permeation chromatography
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Stationary phase: No interactions with analyte.Mobile phase: No interactions with analyte. Analyte must be soluble.Analyte: Analytes must significantly differ in sizeMechanism:• No interactions between analyte, stationary phase and mobile phase• Small analytes can diffuse into pores and therefore take more time to reach the detector• Separation by size
Elution strength of the mobile phase
high elution strength
RP-HPLC NP-HPLC HILIC IC GPC
pola
rity
pola
rity
pola
rity
ioni
c st
reng
th
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low elution strength
pola
rity
pola
rity
pola
rity
ioni
c st
reng
th
Overview important HPLC techniques
HPLC techniqueStationary
phase*Mobile phase* analytes Main interaction
Reversed phase (RP)
C18, C8 modified
SiO2
H2O, methanol, acetonitrile, buffer
salts
Semi polar and non polar
substancesDistribution
Normal phase (NP)
SiO2, Al2O3Hexane, pentane,
benzenePolar substances Adsorption
Ion chromatography (IC)
Polymers with charged
Acidic or alkalineaqueous buffers
Organic and inorganic (an)ions
Electrostatic interactions
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(IC)with charged
moietiesaqueous buffers inorganic (an)ions interactions
Hydrophilic interaction chromatography
(HILIC)
SiO2 and various polar modifications
H2O, methanol, acetonitrile, buffer
saltsPolar substances
Distribution (adsorption)
Gel permeation chromatography
(GPC)Polymers
H2O, organic solvents
Macromolecularsubstances
-
*most commonly used stationary and mobile phases. There are of cause more.
Instrumentation
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Pump and injector
HPLC pumps should be able to generate a continuous, pulsation free flow at a back pressure of up to 400 bar (1000 for UPLC)
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UV/Vis detector, Diodenarray detector
UV/Vis detector
Very similar to UV/Vis spectrometers but different cell geometry. Revisit the UV/Vis spectrometry lecture for the principles of optical spectrometry
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The UV/Vis or diodenarray detector is the standard HPLC detector in routine analysis. It
is suitable to detect the majority of organic substances with an acceptable sensitivity
Diodenarray detector
Fluorescence detector
Excitation: absorption of lightFluorescence: emission of lightNon-radiative relaxation: emission of energy (heat)
Since a portion of the energy is lost as heat the emitted light has a longer wavelength than the absorbed light
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The fluorescence detector is highly selective and sensitive but is limited to fluorescent substances (derivatisation)
Refractive index detector
Detection is based on a different refractive index of the pure solvent and the solvent during elution of a substance.
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The refractive index detector is a universal detector capable of detecting virtually any substance but suffers form poor sensitivity, temperature sensitivity
and is incapable of gradient elution
Evaporative light scattering detector
The column effluent gets nebulized and the mobile phase evaporated, resulting in the formation of analyte particles.
These analyte particles drift through the path of a laser and scatter the laser light. The intensity of the scattered light is
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measured.
All components of the mobile phase must be volatile. Volatile analytes can not be detected!The evaporative light scattering detector
is universal for all non-volatile substances. It is capable of gradient elution but limited to volatile mobile
phase compositions
Mass spectrometer
The analyte has to be ionized and desolvatized. Detection of substances is achieved m/z selective.
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A mass spectrometer is capable of analyzing all ionizable substances with a
high selectivity and sensitivity. It properties are highly dependent on the
instrumental setup.
Various ion sources, mass analyzers and detectors are commercially available and heavily influence the properties and performance of the instrument.
The details of mass spectrometry will be covered in a separate lecture.
Electrical conductivity detector
Measures the conductivity of the column effluent. Changes in the conductivity indicate the presence of eluting ions.
A electrical conductivity detector is most commonly deployed in combination with a suppressor to reduce the
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deployed in combination with a suppressor to reduce the base conductivity of the mobile phase and thus increase the sensitivity
The electrochemical conductivity detector is the standard detector in ion chromatography. It is
selective for charged compounds.
Detectors overview
Detector principle analytes sensitivity selectivity
UV/Vis detector,Diodenarray detector (DAD)
UV/Vis absorption
UV/Vis absorbing substances
0 0
Fluorescence detector (FLD)
fluorescencefluorescent substances
++ ++
Refractive index detector (IRD)
refraction universal -- --
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(IRD)
Evaporative light scattering detector (ELSD)
light scattering
non-volatile substances
- -
Mass spectrometer (MS)*mass
spectrometryionizable substances ++ ++
Electrical conductivity detector (ELCD)
electrical conductivity
ionic substances + -
*content of a separate lecture
Method development – LC technique selection
Selection of a suitable chromatographic technique based on the analyte properties
Semi to non-polar organic substances
RP-HPLC
Semi-polar to polar organic substances
NP-HPLC/HILIC
Ionic substances
IC/HILIC
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While NP-HPLC offers good retention for very polar substances
they are often not well soluble in the deployed apolar organic solvents
Method development – basic screening gradient
Once a chromatographic technique is selected a basic screening gradient should be run(e.g. 95/5 water/methanol to 5/95 water/methanol on a C18 column in case of RP-HPLC)
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Don’t start at 0% water unless the column is compatible with it
Method development – adjustment of slope
The slope of a gradient may heavily influence resolution, peak intensity and analysis time.
A shallow gradient results in an improved resolution at the cost of peak height and time.
A steep gradient increases the
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A steep gradient increases the peak height and shortens the analysis time but resolution may suffer.
In most cases the aim is to achieve a sufficient resolution while maintaining a short analysis time and good peak heights
Mobile phase adjustments – methanol vs. acetonitrile
Viscosity: mixtures of acetonitrile and water have a significantly lower viscosity than mixtures of methanol and water, resulting in a reduced back pressure.
UV-Absorption: methanol (205nm) and acetonitrile (190 nm) have a different UV-cutoff wavelength, resulting in increased sensitivity when using acetonitrile with a UV detector at low wavelengths
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Selectivity: methanol is a protic and acetonitrile a aprotic solvent, resulting in a different selectivity for some analytes (especially analytes with a polar moiety)
Elution strength: methanol and acetonitrile have a similar elution strength in RP-HPLC
Price: methanol is cheaper than acetonitrile
Relative elution strength
Mobile phase adjustments – pH value
The pH value may influence the charge state of a compound.
Ionic compounds are more polar and their non-ionized forms and thus less retained under RP-HPLC conditions. Ionic compounds may interact strongly with silanol groups.
When analyzing ionizable compounds the pH of the mobile phase has to be adjusted
Buffers are effective within a range of± 1 pH around the pKa of their compounds
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Common HPLC buffers are:
Phosphate: pKa 2.1/7.2/12.3 non-volatile
Formate: pKa 3.8 volatile (NH4+ salt)
Acetate: pKa 4.8 volatile (NH4+ salt)
Always consider your detector when selecting a buffer
Dealing with ionic substances
Ionic substances are not well retained in RP-HPLC. There are three ways to deal with them:
1.) Adjustment of pH (not possible for all substances)
2.) Selection of a more suitable chromatographic technique (e.g. IC or HILIC)
3.) Use of ion pair reagents
Ion pair reagents consist of an
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ionic and a hydrophobic part.They pair with the analyte and are retained together with it.
Always consider your detector when selecting an
ion pair reagent
Sample solvent and injection volume
Injecting a sample dissolved in a solvent with a higher elution strength than the mobile phase (starting conditions in case of gradient elution) can result in broad peaks with poor peak shapes and reduced retention times.
This effect is more significant for early eluting peaks and with increased injection volumes.ACN H2O/ACN
70:30
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Always choose a sample solvent that hast a lower elution strength than your
mobile phase
Trends in chromatography – U(H)PLC
U(H)PLC (ultra (high) performance liquid chromatography) is a variant of HPLC with shorter columns (50mm), smaller particles (<2µm), and higher linear velocities.
It allows for faster analysis with reduced solvent consumption at the cost of significantly increased backpressure (instrumentation!)
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Trends in chromatography – solid core particles
Particles are composed of a solid core and a porous shell.Reduced diffusion path lengths (compared to fully porous particles of the same size) result in an decreased influence of mass transfer.Results are increased in a similar was as in UHPLC but without the increase in
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UHPLC but without the increase in backpressure.
Task 1: method development
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Phenol Nitrophenol Anthracene
Suggest a suitable chromatographic method for the quantification of phenol, nitrophenol and anthracene from aqueous samples.Consider the LC technique, the detector and method parameters.
Task 2: method development
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Diquat Diclofenac Anthracene
Suggest a suitable chromatographic method for the quantification of diquat, diclofenac and anthracene from aqueous samples.Consider the LC technique, the detector and method parameters.
Task 3: method optimization
Column: C18 150*4.6mm; 5µmMobile phase: H2O/ACN/H2SO4
75:25:0.03Flow: 1ml/minDetector 1: UV (205 nm)Detector 2: RID
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Your laboratory routinely performs the above mentioned analysis and you are tasked to reduce the cost per analysis without compromising the results. Discuss the usefulness of a) An exchange acetonitrile for methanolb) Gradient elution to reduce the analysis timec) Reduction of the column dimensions (diameter lengths and particle size) and
adjustment the flow rate
Additional information
Book:Douglas A. Skoog, F. James Holler, Stanley R. Crouch: Principles of Instrumental Analysis, 6th Edition, 2007
Internet:http://www.chemguide.co.uk/analysis/chromatogrmenu.html#tophttp://www.sepscience.com/Techniques/LChttp://www.studyhplc.com/novice.php
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Idstein | Köln | Hamburg | Düsseldorf | München | Frankfurt am Main | Berlin | Zwickau | New York