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High performance liquid chromatography Daniel Zahn Idstein | Köln | Hamburg | Düsseldorf | München | Frankfurt am Main | Berlin | Zwickau | New York

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Page 1: High Performance Liquid Chromatography

High performance liquid chromatography

Daniel Zahn

Idstein | Köln | Hamburg | Düsseldorf | München | Frankfurt am Main | Berlin | Zwickau | New York

Page 2: High Performance Liquid Chromatography

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

Page 3: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

• Technique was termed chromatography, derived from the greek word chroma (color) and graphein (to write)

Page 4: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

• Retention by the stationary phase can in most cases be attributed to at least one of the basic mechanisms:

• Adsorption• Distribution

Adsorption

Distribution

Page 5: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

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

Page 6: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

• 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

Page 7: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

• 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

Page 8: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

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

Page 9: High Performance Liquid Chromatography

Peak types

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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Page 10: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

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.

Page 11: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

number (N)

l column length

The plate number significantly influences

the broadness of a peak

Page 12: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

mobile phase flowrate u

optimal flowrate

minimal plate height

A eddy diffusion

B longitudinal diffusion

C mass transfer

Page 13: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

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

Page 14: High Performance Liquid Chromatography

Van-Deemter-equation

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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Page 15: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Baseline separation is mandatory if quantification

is performed with non-selective detectors!

Page 16: High Performance Liquid Chromatography

Optimization of resolution

The three important factors for the resolution are:

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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

resolution are:

• the efficiency

• the retention factors

• and the selectivity

Page 17: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

• increasing the column length (also increases analysis time!)

• deploying smaller particles (also increases backpressure!)

• optimization of the flow rate

• other factors

Page 18: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

almost no influence on the resolution

The retention factor can be improved by:

• adjusting the solvent strength (effective and simple!)

• changing the stationary phase

Page 19: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

• 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.

Page 20: High Performance Liquid Chromatography

Reversed Phase HPLC

OH2

OH2

OH2

OH2

OH2

OHOH2

OH2

OH2

OH2

NCH3

NCH3NCH3

NCH3

NCH3

NCH3

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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

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

Page 21: High Performance Liquid Chromatography

Normal Phase-HPLC

CH3 O

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

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

Page 22: High Performance Liquid Chromatography

Ion chromatography

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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

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

Page 23: High Performance Liquid Chromatography

Hydrophilic interaction liquid chromatograph

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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

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

Page 24: High Performance Liquid Chromatography

Gel permeation chromatography

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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

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

Page 25: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

low elution strength

pola

rity

pola

rity

pola

rity

ioni

c st

reng

th

Page 26: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

(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.

Page 27: High Performance Liquid Chromatography

Instrumentation

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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Page 28: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Page 29: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

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

Page 30: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

The fluorescence detector is highly selective and sensitive but is limited to fluorescent substances (derivatisation)

Page 31: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

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

Page 32: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

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

Page 33: High Performance Liquid Chromatography

Mass spectrometer

The analyte has to be ionized and desolvatized. Detection of substances is achieved m/z selective.

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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

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.

Page 34: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

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.

Page 35: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

(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

Page 36: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

While NP-HPLC offers good retention for very polar substances

they are often not well soluble in the deployed apolar organic solvents

Page 37: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Don’t start at 0% water unless the column is compatible with it

Page 38: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

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

Page 39: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

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

Page 40: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

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

Page 41: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

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

Page 42: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Always choose a sample solvent that hast a lower elution strength than your

mobile phase

Page 43: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Page 44: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

UHPLC but without the increase in backpressure.

Page 45: High Performance Liquid Chromatography

Task 1: method development

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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

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.

Page 46: High Performance Liquid Chromatography

Task 2: method development

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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

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.

Page 47: High Performance Liquid Chromatography

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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Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

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

Page 48: High Performance Liquid Chromatography

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