Essentials in Modern HPLC Separations

532 Pages·2012·9.286 MB·English

by  Serban Moldoveanu and Victor David (Eds.)

ESSENTIALS IN
MODERN HPLC
SEPARATIONS
S C. M 1, V D 2
ERBAN OLDOVEANU ICTOR AVID
1R.J.ReynoldsTobaccoCo.,Winston-Salem,NC,USA
2UniversityofBucharest,Bucharest,Romania
AMSTERDAM(cid:129)BOSTON(cid:129)HEIDELBERG(cid:129)LONDON(cid:129)NEWYORK(cid:129)OXFORD
PARIS(cid:129)SANDIEGO(cid:129)SANFRANCISCO(cid:129)SYDNEY(cid:129)TOKYO
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Moldoveanu,Serban.
EssentialsinmodernHPLCseparations/SerbanC.Moldoveanu,VictorDavid.
p.cm.
Includesbibliographicalreferencesandindex.
ISBN978-0-12-385013-3
1.Separation(Technology)2.Highperformanceliquidchromatography.I.David,Victor,1955-II.Title.
TP156.S45M652013
660’.2842–dc23
2012016476
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AcataloguerecordforthisbookisavailablefromtheBritishLibrary
Theauthorsarepersonallyresponsibleforthecontent,accuracy,andconclusionsofthisbook.
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In memory of Professor Candin Liteanu,pioneer in implementing anddeveloping
high performance liquid chromatography in Romania
Preface
Onemayask“whyanotherbookonHPLC?” can be downloaded (free) from http://www.
Thefieldisrapidlyevolving,andnewinforma- epa.gov/oppt/exposure/pubs/episuite.htm.
tion is being accumulated from a large number Themaingoalofthebookistoprovidemate-
of original studies published in scientific and rial thatdescribesusefulinformationregarding
technical journals but not reviewed yet in HPLC.Thechallengeinmakingsuchapresenta-
a book. From time to time, this information tion is considerable, and the authors took
needs to be collected, classified, and presented advantage of the information from a number
systematically. This new book describes of other books available on the market. Among
a number of such developments that more such books are Introduction to Modern Liquid
recently started to be utilized. One additional Chromatography (L. R. Snyder, J. J. Kirkland,
reason for a new book is that utilization of J. W. Dolan, Wiley, 2010), HPLC for Pharmaceu-
HPLC is widespread, and a large number of ticalScientists(YKazakevich,R.LoBrutto,Wiley,
readers may have different needs and interests 2007), HPLC Columns, Theory, Technology, and
that are not completely addressed in other Practice (U. D. Neue, Wiley, 1997), and Practical
books. The main purpose of the present book High-Performance Liquid Chromatography (V. R.
istoprovidepractical guidanceinthe selection Meyer, Wiley, 2010). An enormous number of
ofcolumns,ofmobilephases,andofseparation applications of HPLC have been published in
conditionsfordifferenttypesofHPLC,together peer-reviewed journals, in a number of books,
withjustifyingwhyaparticularselectionisrec- and on the web. These sources of information
ommended. Another purpose is to provide are considered more useful for finding direct
criteria for selecting specific HPLC methods. applications as compared to a new book with
For example, the present volume shows how a limited number of pages. For this reason,
the octanol/water partition coefficient (log except for examples, the present book does not
K ) of the analyte can be a very useful param- contain recipesfor particular analyses.
ow
eter for chromatographers. Discussions This book starts with an introduction that
regarding the use of this parameter in HPLC provides basic information about HPLC and
have been previously published, but this book HPLC instrumentation. The next chapter
applies it consistently. Octanol/water partition describes common parameters used for charac-
coefficients for many molecules are readily terization of an HPLC separation; Chapter 3 is
available and are extensively used in the phar- dedicated to equilibria in HPLC; Chapter 4
maceutical field as well as for description of discusses interactions at the molecular level
the environmental fate of compounds. A pro- that take place during different types of HPLC
gram available from the U.S. Environmental separations;andChapter5examinesthesepara-
ProtectionAgency(EPA)containingbothadata- tionmechanismsindifferentHPLCtypes.Inthe
basewithexperimentallogK valuesformany following chapters, the material shifts toward
ow
chemicalsandaprogramforestimatinglogK direct applications and covers columns and
ow
xv
xvi
PREFACE
mobilephasesinHPLC,aswellasthecharacter- types has therefore been approached, which is
ization of analytes that determines the HPLC believed to be easier to follow. The authors
selection. The last chapter is dedicated to the wish to thank the editorial team from Elsevier,
practiceofHPLCseparations.Whilemostbooks Linda Versteeg, Jill Cetel, Beth Campbell,
onHPLCfocusthepresentationonthetypesof and Mohanapriyan Rajendran, for their con-
liquidchromatography(reversedphase,normal tribution to the publication of this book.
phase,ionexchange,etc.),thisbookisorganized Also, the authors express their thanks to Paul
based on the view that there are significant Braxton,CarolMoldoveanu,andMichaelDavis
unifying points among all HPLC types. A for reviewing the manuscript and suggesting
more uniform presentation including all HPLC valuable corrections.
C H A P T E R
1
Basic Information about HPLC
O U T L I N E
1.1. Introductionto HPLC 2 1.4. Overview ofHPLC Instrumentation 20
What is Chromatography? 2 GeneralComments 20
Types of Equilibria inHPLC 3 SchematicDescription of an HPLC
Criteria forthe Classification of HPLC Instrument 20
Procedures 6 Solvent SupplySystems 20
Role of Polarity inHPLC 7 Pumping Systems 22
Qualitative Analysis and HPLC Main Injectors 27
Useas aQuantitative Analytical Tubing and Connectors 28
Technique 8 Chromatographic Columns 29
Setups forMultidimensional Separations 31
1.2. Main Types ofHPLC 9
Other Devicesthat are Part of the HPLC
AClassification of HPLC Types 9
System 31
Relation between the Type of HPLC
GeneralCommentson Detectors 33
and Equilibrium Mechanism 14
SpectrophotometricDetectors 34
1.3. Practice ofHPLC 14 Fluorescence and Chemiluminescence
GeneralAspects 14 Detectors 36
Selection of the Type of HPLC RefractiveIndexDetectors 38
fora ParticularApplication 15 Electrochemical Detectors 39
Sample Collection and Sample Preparation Mass Spectrometric Detectors 42
forHPLC 15 Other Types of Detectors 46
Injection 16 Selection of aDetector for theHPLC
Column Selection inHPLC 17 Separation 47
Mobile Phase Selection 17 Fraction Collectors 50
Detection inHPLC and Quantitation Controlling and Data Processing Units 50
Procedures 18
EssentialsinModernHPLCSeparations
1
http://dx.doi.org/10.1016/B978-0-12-385013-3.00001-X Copyright(cid:1)2013ElsevierInc.Allrightsreserved.
2
1. BASICINFORMATIONABOUTHPLC
1.1. INTRODUCTION TO HPLC a column packed with very small porous parti-
cles(1e5mmindiameter),andtheliquidmobile
phase (or eluent) is moved through the column
What is Chromatography?
by a pump (at elevated pressure). Solutes are
The term chromatography designates several injected in the mobile phase as a small volume
similar techniques that allow the separation of at the head of the chromatographic column.
different molecular species from a mixture. A schematic diagram of the separation process
Applications of chromatography are numerous is shown in Figure1.1.1.
and can be related to laboratory or industrial As the mobile phase flows, the eluted mole-
practices. The molecular species subjected to culesthatareexitingthecolumncanbedetected
separation exist in a sample that is made of by various procedures. The eluted molecules
analytes and matrix. Theanalytes arethe molec- differ from the mobile phase components
ularspeciesofinterest,andthematrixistherest by certain physicochemical properties (UV-
ofthecomponentsinthesample.Forchromato- absorption, refractive index, fluorescence,
graphic separation, the sample is introduced in molecular mass and fragmentation in a mass
a flowing mobile phase that passes a stationary spectrometer, or others), which make them
phase. The stationary phase retains stronger or detectable. Finally, an electrical signal is typi-
weaker different passing molecular species cally associated with molecular detection, and
and releases them separately in time, back into the graphic output of this signal is known as
the mobile phase. When the mobile phase is a chromatogram. The separated components of
a gas, the chromatography is indicated as gas a mixture eluting at different times (known as
chromatography (GC), and when it is a liquid, retention times t ) are displayed as peaks in the
R
it is indicated as liquid chromatography (LC). chromatogram. Different peaks (or patterns)
Other types of chromatography include super- on the chromatogram belong to different
critical fluid, countercurrent, and electrochro- components of the separated mixture. An
matography. When the sample is present as example of a chromatogram with the retention
a solution, its components are indicated as times written above the peaks is shown in
solutes. Sample dissolution and/or preliminary Figure1.1.2.Asshowninthisfigure,thesepara-
modifications are frequently necessary to have tion of the peaks can be very good or only
the analytes amenable for a chromatographic partial. Also, some compounds may not be
separation (see, e.g., [1]). In high performance separated at all. Separated peaks may indicate
(or pressure) liquid chromatography (HPLC), individual compounds only when each peak
the stationary phase is typically in the form of corresponds to a single molecular species.
Mixed molecular species Separated
previously injected molecular species
Flow Flow
Time 2
Time 1
Stationary phase Stationary phase
FIGURE1.1.1 Simplifiedillustrationoftheseparationprocessinchromatography(theblackandwhitestarsindicatetwo
differentmolecularspecies).
3
1.1. INTRODUCTIONTOHPLC
Intensity units
600
1.13
500
400 5.12
300
200
3.03 4.33
4.61
100
0
1.0 2.0 3.0 4.0 5.0 6.0
Time min -->
FIGURE1.1.2 Pictureofachromatogramindicatingtheretentiontimeforsomeofthepeaks.
In HPLC the analytes are separated from each analytes, the separation process is achieved
other and from the matrix as well as possible. depending on the choice of a chromatographic
The zones occupied by a specific analyte when column and a specific mobile phase. The detec-
it is eluted from the chromatographic column tion step is achieved using one or more detec-
(peak width in a chromatogram) can be nar- tors,andthesensitivity,selectivity,andstability
rowerorwider.Thewidthofthesezonesaffects of these detectors is essential for the success of
the separation, and for two analytes with the HPLC analysis. The present text does not
differentretentiontimes,theseparationisbetter include a detailed discussion of detection and
when the elution zones arenarrower. measurement of the analytes, and focuses
The peaks in the chromatogram may have mainlyontheirseparation.
different heights (and peak areas) depending Separation by HPLC can also be used for
on a number of factors such as the amount of semipreparative or preparative purposes, some
compound in the mixture, amount of sample with industrial applications. In this case, the
injected, and sensitivity of the detection proce- separated compounds of interest are collected
dure. Since peak areas are dependent on the forfurtherutilization.However,themainfocus
amount of the compound, HPLC can be used of the present volume is analytical HPLC, and
for quantitation after a proper calibration. In semipreparative and preparative HPLC are
this way, HPLC became an excellent technique beyond the scope ofthis work.
for separation and quantitation of compounds
eveninverycomplexmixturesandiscurrently
Types of Equilibria in HPLC
the most widely used analytical technique ever
practiced. The separation process in HPLC is based on
As the previous short description of HPLC an equilibrium established between the mole-
shows, the technique has two distinct parts: cules present in the mobile phase and those
(1) separation of the analytes andof the matrix, retained in the stationary phase. The difference
(2) detection and measurement of the analytes. in the concentration of a molecular species in
Thediscussionabouttheseparationisthemain one phase and in another determines whether
subject of this book. Based on the nature of the thespeciesisretainedorelutedwiththemobile
4
1. BASICINFORMATIONABOUTHPLC
Mobile phase
phase. When the concentration of the solute
(analyte) is higher in the mobile phase than in Analyte ● ● ●
the stationary phase, the solute is eluted faster ● ● ● ● ● ●
●
fsriotemhtahpepcehnrsomwahteongrathpehiccocnocleunmtrna.tiTohneoofppthoe- staLtiqiounidary ● ● ● ● ● ●
phase ● ●
solute is higher in the stationary phase. In this ●
case, the solute is more strongly retained and ● ●
the elution takes place after a longer period of Solid support
time.
Common types of equilibria for a molecular FIGURE 1.1.3 Schematic description of partition
equilibrium.
speciesbetweentwophasesinclude,forexample,
the distribution of a compound between two
immisciblesolvents.Anothercommontypetakes
phases. A schematic description of the parti-
place during the retention of a compound from
tion chromatography process is shown in
afluidonanadsorbingmaterialsuchascharcoal.
Figure1.1.3.Inpartitionchromatography,the
Chemicalequilibriuminasolution,forexample,
concept of “immobile liquid” is commonly
between twoionic compounds, is also a known
approachedina“loose”manner.Forexample,
type. The main types of equilibria encountered
a layer of adsorbed water on the surface of
in chromatography can be summarized as
a silica solid support, or a layer of bonded
follows:
organic chains on a silica surface (such as in
1) Partition equilibrium. This type of equilibrium the common C18 chromatographic columns),
takes place when the molecules of the solute or a layer of mechanically held polymer on
aredistributedbetweentwoliquidphases.In aninertcoreareallconsideredliquidstation-
HPLC, one liquid phase is kept immobile on aryphasesforpartitionchromatography.The
a solid material, and the other is mobile (the possibility of performing chromatography
eluent). The immobilization of the liquid to using two liquid phases without having one
becomeastationaryphaseinpartitionchroma- liquidphaseimmobilizedisexploitedincoun-
tography is achieved, for example, when the tercurrent chromatography. However, this
liquid is highly polar and can establish subject is beyond the purpose of the present
hydrogen bonds with the solid support. One book(fordetailssee,e.g.,[2]).
such example is water on a silica surface. In 2) Adsorptionequilibrium.Thistypeofequilibrium
this case, the mobile phase should consist of takes place when molecules are exchanged
a liquid less polar than water. However, the between a solid surface and a liquid mobile
partition equilibrium can also be applied for phase. Assuming that the stationary phase is
anonpolarstationaryphaseandamorepolar verypolarcomparedtothemobilephase,the
mobile phase. The theory of separation in polar molecules from the mobile phase are
partitionchromatographyisbasedonliquid/ adsorbedonthesolidstationaryphasesurface,
liquid extraction principles. The different whilethelesspolarmoleculesarekeptmainly
molecular species, being in continuous equi- in the mobile phase. Being in equilibrium
librium between the mobile and stationary between the solid and the liquid, the more
phase, will be separated based on their polarmoleculesalsoelutefromthechromato-
tendency to exist in higher concentration in graphic column, but later than the less polar
the mobile liquid or in the stationary liquid, compounds. A schematic description of the
in accordance with their affinity for these adsorptionchromatographyprocessisshown
5
1.1. INTRODUCTIONTOHPLC
Mobile phase Mobile phase
Analyte ● ● ● ●● ● ● aInoanlyicte
● ●
●
●
● ● ● ●
●
●
●
●
● ●
Solid support acting Stationary phase with
as stationary phase covalently bound ionic groups
FIGURE 1.1.4 Schematic description of adsorption FIGURE 1.1.5 Schematic description of ion exchange
equilibrium. proces.
inFigure 1.1.4. The partition and the adsorp- a porous structurein which small molecules
tion are utilized basically as models for can penetrate and spend time passing
describingthetypeofequilibrium,butadiffer- throughthe long channelsof the solid mate-
ence between the two processes is not com- rial, while large molecules cannot penetrate
monlyapparentfromathermodynamicpoint the pore system of the stationary phase and
ofview[3].Also,inmanyinstancesthesepara- arenotretained.AppliedinHPLC,thelarge
tioncanbeviewedeitherasapartitionorasan moleculeseluteearlier,whilethesmallmole-
adsorption, the differentiation being made culesareretainedlonger.Anequilibriumcan
onlywiththepurposeofestimatingdifferently be envisioned between molecules in the
theseparationparameters,whiletheclassifica- mobile phase and those partly trapped in
tionhasnoeffectontherealprocess. the solid matrix. A schematic description of
3) Equilibria involving ions. Equilibria between the size exclusion process is shown in
ions in solutions take place in numerous Figure1.1.6.
chemical reactions. For applications in 5) Affinityinteractions.Thistypeofinteractionis
HPLC, one ionic species must be immobi- typicalforproteinbindingandleadstoequi-
lized, for example, by being connected libria that allow very specific separations.
through a covalent bond to a solid matrix. Examples of such interactions are protein-
One example of this type of ion can be
a sulfonic group connected to polystyrene.
Mobile phase
The ions in solution can be bound by ionic Small
molecule
interactions to the immobilized counterion
analyte ●
or may remain in solution. The equilibrium ●
between solid phase and mobile phase, ●
Large
● ●
depending on the strength of the bond molecule
● ● ●
to the stationary phase, may provide analyte ● ● ● ● ● ●
●
a means for separation. A schematic ●
●
description of the interactions in the ion-
Porous gel
exchangechromatographyprocessisshown
stationary phase
in Figure 1.1.5.
4) Equilibria based on size exclusion. Size exclu- FIGURE 1.1.6 Schematic description of size exclusion
sion uses a stationary phase that consists of process.