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ESSENTIALS IN
MODERN HPLC
SEPARATIONS
SECOND EDITION
Serban Moldoveanu
Senior Principal Scientist, RJ Reynolds Tobacco Co., Winston-Salem, NC, United States
Victor David
Professor and Head of the Department of Analytical Chemistry, University of Bucharest, Bucharest, Romania
Preface
High-performance liquid chromatography From the first edition, the book was subject to
(HPLC) is the most utilized technique in chemical a significant number of changes. New literature
analysis of a variety of materials. As a result, a very sources were used, new references were added,
large amount of information is available for HPLC and also the initial structure of the book was
in peer-reviewed journals, in books, in manufac- modified to make it more useful for practical
turer catalogues, and on the internet. Besides the applications. The book was organized in four
applications of HPLC for analytical purposes, parts, basic information, main types of HPLC
there is also interest in using HPLC for the estima- separations, practice of HPLC analysis, and
tion of various physico-chemical characteristics of appendices. The large tables containing detailed
molecules and for the description of certain data on the main commercial HPLC stationary
processes which can be studied with difficulty phases and columns and on main properties of
by other means. The goal of present book is to offer solvents used in the mobile phases for HPLC
a coherent synthesis of the large amount of infor- were placed as appendix to avoid breaking the
mation about HPLC and to present the novel text flow. This new edition of the book was
developments in the field. Separation is a key written with the purpose to offer both a simple
part of HPLC and the book is focused on the theoretical background about HPLC and guid-
understanding of various aspects of it such as ance for the utilization of different types of
the distribution process between nonmiscible HPLC in practice. No explicit methods for the
phases, the molecular aspects involved in the analysis of specific analytes are described in
separation, the parameters that characterize the the book (except for some examples), this field
separation, the types and performances of mate- being much better covered in dedicated peer-
rials used in the construction of chromatographic reviewed literature.
HPLC columns that provide the physical support The authors are indebted to the editorial team
for the separation, the role of mobile phase in the Mrs. Kathryn Eryilmaz, Ms. Emerald Li, and
separations, etc. The material is presented having Mr. Rajan Vijay Bharath from Elsevier, for their
in mind the applicability of theory for the selection assistance in publishing this book. Also, we are
of the best HPLC analytical procedure, and thankful to the referees of this new edition
besides the separation, other aspects of HPLC for their fair comments and useful
are also included such as instrumentation and recommendations.
utilization in practice of HPLC.
ix
Book • Second Edition • 2022

Authors:
Serban Moldoveanu and Victor David
Table of contents
Front Matter, Copyright, Preface
Section I: Basic information about HPLC
Book chapter Abstract only

Chapter 1 - Introductory information regarding HPLC
Pages 3-20
View abstract

Chapter 2 - Overview of HPLC instrumentation and its use
Pages 21-61
View abstract

Chapter 3 - Parameters for the characterization of HPLC separation
Pages 63-105
View abstract

Chapter 4 - Equilibrium types in HPLC
Pages 107-146
View abstract

Chapter 5 - Intermolecular interactions
Pages 147-177
View abstract

Chapter 6 - Characterization of analytes and matrices
Pages 179-205
View abstract

Chapter 7 - Mobile phases and their properties
Pages 207-269
View abstract
Essentials in Modern HPLC Separations | ScienceDirect https://www.sciencedirect.com/book/9780323911771/essentials-in-m...

Chapter 8 - Analytical HPLC columns and their characteristics
Pages 271-337
View abstract
Section II: Main types of HPLC separations

Chapter 9 - Reversed-phase HPLC
Pages 341-419
View abstract

Chapter 10 - Other HPLC separations performed on hydrophobic stationary phases
Pages 421-446
View abstract

Chapter 11 - Hydrophilic interaction liquid chromatography
Pages 447-477
View abstract

Chapter 12 - Other HPLC separations performed on polar stationary phases
Pages 479-484
View abstract

Chapter 13 - Ion exchange, ion-moderated, and ligand exchange liquid chromatography
Pages 485-512
View abstract

Chapter 14 - Chiral HPLC separations
Pages 513-539
View abstract

Chapter 15 - Size exclusion HPLC
Pages 541-558
View abstract

Chapter 16 - Affinity, immunoaffinity, and aptamer type HPLC
Pages 559-569
View abstract

Chapter 17 - Mixed-mode HPLC
Pages 571-576
View abstract
Section III: Practice of HPLC analysis

Chapter 18 - Utilization of HPLC in chemical analysis
Pages 579-594
View abstract
Book chapter No access

Appendix to Chapter 6
Pages 595-597
2 de 4 12/09/2022, 13:31
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Appendix to chapter 7
Pages 599-613

Pages 615-620

Pages 621-668

Pages 669-674

Pages 675-682

Pages 683-688

Pages 689-692
Book chapter Full text access

Index
Pages 693-705
C H A P T E R
1
Introductory information regarding HPLC
1.1 Preliminary discussion about HPLC (GC)). For these reasons, HPLC is an ideal
analytical technique for many types of samples
General comments regardless how complex or simple they are.
The steps in a chemical analysis involve both
conceptual activities and also experimental
What is chromatography and what is
ones and they can be schematically summarized
HPLC?
as shown in Fig. 1.1.1 [1].
In the chain of activities indicated in Fig. 1.1.1, The term chromatography designates several
the most common core analysis, in current prac- similar techniques that allow the separation of
tice, is high-performance liquid chromatography different molecular species from a mixture. Ap-
(HPLC). The main advantages of HPLC as a plications of chromatography are numerous,
method of analysis include the combination of and can be related to laboratory or industrial
a powerful separation step (that is presented in practices. Analytical chromatography uses a
detail in this book) with a sensitive measurement chromatographic separation of the compound
capability, plus the possibility to be applied to a from a sample and for the identification and/or
wide variety of molecules regardless of molecu- measurement of these compounds uses a
lar weight of the analytes or of their volatility specific detection. The molecular species from
(required, for example, in gas chromatography the sample are indicated as analytes and matrix.
Collection of Results
information interpretation
Data
Planning processing
Sample Sample Sample Core

collection conservation preparation analysis
FIGURE 1.1.1 Steps in a chemical analysis (conceptual steps in dotted frame).

https://doi.org/10.1016/B978-0-323-91177-1.00006-5 3 © 2022 Elsevier Inc. All rights reserved.
4 1. Introductory information regarding HPLC
The analytes are the molecular species of interest diagram of the separation process is shown in
and the matrix forms the rest of components in Fig. 1.1.2.
the sample. For chromatographic separation, The eluted molecules from the chromato-
the sample components are introduced in a flow- graphic column differ from the mobile phase
ing mobile phase that passes a stationary phase. The components by certain physico-chemical proper-
stationary phase retains stronger or weaker ties, which makes them detectable. Common
different passing molecular species and releases properties used for detection in liquid chroma-
them separately in time back into the mobile tography are the density, the UV absorption,
phase. When the mobile phase is a gas, the chro- the fluorescence, the type of ions in a mass spec-
matography is indicated as gas chromatography trometer, etc. The role of the detector in the chro-
(GC), and when it is a liquid, is indicated as matographic analysis is to produce an electrical
liquid chromatography (LC). Other types of signal which depends on the specific physico-
chromatography are known and they include chemical property of the material eluting from
supercritical fluid, countercurrent, electro- the chromatographic column. The electrical
chromatography, etc. When the sample is pre- signal caused by the detection of specific molec-
sent as a solution its components are indicated ular species along a chromatographic separation
as solutes. Sample dissolution and/or prelimi- (chromatographic run) is translated into a
nary modifications are frequently necessary to graphic output which is known as a chromato-
have the analytes amenable for a chromatogram. The separated components of a mixture
graphic separation (e.g., Ref. [1]). In a common eluting at different times (known as retention
type of liquid chromatography, the stationary times tR) are displayed as peaks in the chromato-
phase is in the form of a column packed with gram. Due to diffusion (and other effects) in the
very small porous particles (1.7e10.0 mm in chromatographic column, the peaks have ideally
diameter), or a porous monolithic material, and a Gaussian shape. Different peaks (or patterns)
the liquid mobile phase (or eluent) is moved on the chromatogram belong to different compo-
through the column by a pump (at elevated pres- nents of the separated mixture. An example of an
sure). This type of chromatography is indicated HPLC chromatogram obtained from a standard
as high performance (or pressure) liquid chro- mixture of organic acids separated on a Synergi
matography (HPLC). In HPLC, the sample in 4u Hydro-RP column with the retention times
liquid form (solution) is injected in the mobile written above the peaks is shown in Fig. 1.1.3 [2].
phase as a small volume at the head of the chro- As shown in Fig. 1.1.3, the separation of the
matographic column. As the mobile phase flows, peaks can be very good but also can be only par-
the sample constituents are separated and the tial. Some compounds may not be separated at
eluted molecules that exit the column can be all. Separated peaks may indicate individual
detected by various techniques. A schematic compounds only when each peak corresponds
FIGURE 1.1.2 Schematic illustration of the separation process in chromatography showing the initial time (Time 1) and
after the compounds started to be separated (Time 2) (the black and white stars indicate two different molecular species).
I. Basic information about HPLC

1.1 Preliminary discussion about HPLC 5
FIGURE 1.1.3 Picture of an HPLC chromatogram indicating the retention time for some of the peaks in a standard mixture
of organic acids [2].
to a single molecular species. In HPLC, the analy- mixtures and is currently the most widely used
tes should be separated from each other and analytical technique ever practiced.
from the matrix, as well as possible. For example, It can be seen from the previous short descrip-
in the chromatogram from Fig. 1.1.3, the first tion of HPLC, that the technique has two distinct
peaks belong to the separated matrix (their reten- parts: (1) separation of the analytes and of the
tion time is not indicated). The zones occupied by matrix, (2) detection and measurement of the
a specific analyte when it is eluted from the chro- analytes. The discussion about the separation is
matographic column (peak width in a chromato- the main subject of this book. Based on the na-
gram) can be narrower or wider. The width of ture of the analytes, the separation process is
these zones affects the separation, and for two achieved depending on the choice of a chromato-
analytes with different retention times, the separa- graphic column and a specific mobile phase. The
tion is better when the elution zones are narrower. detection step is achieved using one or more de-
The peaks in the chromatogram may have tectors, and the sensitivity, selectivity, and stabil-
different heights (and peak areas) depending ity of these detectors are essential for the success
on a number of factors such as the amount of of the HPLC analysis. Present book does not
compound in the mixture, amount of sample include a detailed discussion on detection and
injected, and sensitivity of the detection proced- measurement of the analytes, and is focused
ure. Since peak areas are dependent on the mainly on their separation.
amount of the compound injected into the col- Separation by HPLC can also be used for
umn, HPLC can be used for quantitation after a semipreparative or preparative purposes, some
proper calibration. In this way, HPLC became with industrial applications. They differ to the
an excellent technique for separation and quanti- analytical HPLC by the experimental design,
tation of compounds even in very complex mainly by using columns with larger dimensions

(length, diameter, and particle size that offer Chemical equilibrium in a solution, e.g., between
lower back pressures than analytical HPLC) two ionic compounds, is also a known equilib-
and by the scale of samples, in order to isolate, rium type, although its classification refers
collect, enrich, or purify the components of inter- more to the forces involved in the separation.
est from higher amounts of samples. For A summary of main types of equilibria encoun-
example, the analytical columns packed with tered in chromatography is given below:
stationary phase particle sizes of 3e5 mm, while
(1) Partition equilibrium. This type of equilibrium
for preparative columns the particle size are
takes place when the molecules of the solute are
larger than 10 mm. The separated compounds
distributed between two liquid phases. In
of interest are collected and possibly object of
HPLC, one liquid phase is kept immobile on a
further purification [3]. However, the main focus
solid material, and the other is mobile (the
of this book is analytical HPLC, and semiprepar-
eluent). The immobilization of the liquid to
ative and preparative HPLC are beyond the
become a stationary phase in partition
scope of the present material.
chromatography is achieved, for example, when
the liquid is highly polar and can establish
hydrogen bonds with the solid support, which is
Types of equilibria in HPLC also polar. One such example is water on a silica
surface. In this case, the mobile phase should
The separation process in HPLC is based on
consist of a liquid less polar than water.
an equilibrium established between the mole-
However, the partition equilibrium can also be
cules present in the mobile phase and those
applied for a nonpolar stationary phase and a
retained in the stationary phase. The difference
more polar mobile phase. The theory of
in the concentration of a molecular species in
separation in partition chromatography is based
one phase and in another determines if the spe-
on liquid/liquid extraction (LLE) principles. The
cies is retained or eluted with the mobile phase.
different molecular species, being in continuous
When the concentration of the solute (analyte)
equilibrium between the mobile and stationary
is higher in the mobile phase than in the station-
phase, will be separated based on their tendency
ary phase, the solute is eluted faster from the
to exist in higher concentration in the mobile
chromatographic column. The opposite happens
liquid or in the stationary liquid, in accordance
when the concentration of the solute is higher in
with their affinity for these phases. A schematic
the stationary phase. In this case, the solute is
description of the partition chromatography
more strongly retained and the elution takes
process is shown in Fig. 1.1.4. In partition
place after a longer period of time.
chromatography, the concept of “immobile
Common types of equilibria for a molecular
species between two phases include, for
example, the distribution of a compound be-
tween two immiscible solvents (partition equilib-
rium). Another common type takes place during
the retention of a compound from a fluid on an
adsorbing material such as porous graphitic car-
bon. In a separation, it is possible that more than
one type of equilibrium takes place although
some equilibria can be characterized as mainly
partition or mainly adsorption. Not all equilibria FIGURE 1.1.4 Schematic description of partition
can be classified as partition or adsorption. equilibrium.

liquid” is commonly approached in a “loose”
manner. For example, a layer of adsorbed water
on the surface of a silica solid support, or a layer
of bonded organic moieties on a silica surface
(such as in the common C18 chromatographic
columns), or a layer of mechanically held
polymer on an inert core are all considered
liquid stationary phases for partition
chromatography. The types of interactions in
partition equilibria can be of different types FIGURE 1.1.5 Schematic description of adsorption
equilibrium.
including hydrophobic interactions, van der
Waals interactions, ionic interactions, etc.
The possibility of performing (3) Equilibria involving ions. Equilibria between
chromatography using two liquid phases ions in solutions take place in numerous
without having one liquid phase immobilized is chemical reactions. For applications in HPLC,
exploited in countercurrent chromatography one ionic species must be immobilized, for
[4,5]. This subject is beyond the purpose of this example, by being connected through a
book (for details, see e.g., Ref. [6]). covalent bond to a solid matrix. An example of
this type of ion can be a sulfonic group
(2) Adsorption equilibrium. This type of
connected to polystyrene. The ions from
equilibrium takes place when molecules are
solution can be bound by ionic interactions to
exchanged between a solid surface and a liquid
the immobilized counterion or may remain in
mobile phase. For example, carbon black can
solution. The equilibrium between solid
adsorb nonpolar molecules on the solid
phase and mobile phase, depending on the
stationary phase surface, while the more polar
strength of the bond to the stationary phase,
molecules are kept mainly in the mobile phase.
may provide a means for separation. This type
Being in equilibrium between the solid and the
of interaction is usually indicated as ion
liquid, the nonpolar molecules also elute from
exchange. Although equilibria involving ions
the chromatographic column, but later than the
appear to be more referring to the type of
polar compounds. A schematic description of
interaction, the separation involving ion
the adsorption chromatography process is
exchange is different from partition or
shown in Fig. 1.1.5.
adsorption. A schematic description of the
The partition and the adsorption are utilized
interactions in ion exchange chromatography
basically as models for describing the type of
process is shown in Fig. 1.1.6.
equilibrium, but a difference between the two
processes is not commonly apparent from (4) Equilibria based on size exclusion. Size
thermodynamic point of view [7]. Also, in many exclusion uses a stationary phase that consists of
instances, the separation can be viewed either as a porous structure in which small molecules can
a partition or as an adsorption, the penetrate and spend time passing through the
differentiation being made only with the long channels of the solid material, while large
purpose to estimate differently the separation molecules cannot penetrate the pore system of
parameters, while the classification has no effect the stationary phase and are not retained.
on the real process that may take place with both Applied in HPLC, the large molecules elute
types of equilibrium. earlier, while the small molecules are retained

Role of polarity in HPLC

One common concept related to HPLC sepa-
rations is that of “polarity.” Polarity refers to an
asymmetrical charge distribution in a molecule,
which causes the molecule to act as an electric
dipole. However, charge distribution is a very
complex concept, and the calculation of the
values for charge density, used to characterize
FIGURE 1.1.6 Schematic description of ion exchange charge distribution in a molecule, is usually a
process. difficult task. Also, polarity can refer to the ana-
lytes, to the mobile phase or to the stationary
phase. The compounds are typically indicated
longer. An equilibrium can be envisioned as polar when opposite partial charges are
between molecules in the mobile phase and known to be present in the molecule, when spe-
those partly trapped in the solid matrix. A cific physical properties such as water solubility
schematic description of size exclusion process is or solubility on solvents miscible with water
shown in Fig. 1.1.7. are known, or when specific functional
As in the case of other types of separation, groups known to be “polar” such as eCOOH,
size exclusion process is frequently associated or eNH2 are present in the molecule. In addition
with other type of equilibrium than partition or to that, during molecular interactions, the charge
adsorption. distribution of the molecules suffers changes
(5) Equilibria based on affinity interactions. This (expressed by polarizability). In any interaction,
type of interaction is also more referring to the the polarizability affects the charge distribution
interaction types and is typical for protein of the molecules. For these reasons, comparing
binding. This type of equilibria allows very molecules or phases as “more polar” or “less po-
specific separations. Examples of such lar” is not a quantitative assessment. The oppo-
interactions are proteineantibody, site to the polar character is the hydrophobic
avidinebiotin, etc. Affinity chromatography is character (or lipophilic character; the two terms
widely used at low pressure for protein are not interchangeable, hydrophobicity being
purification, but is also applied in HPLC. the physical property of a molecule to be
repelled from water, while lipophilicity referring
to the ability of a chemical compound to dissolve
in fats, oils, lipids, and nonpolar solvents).
Nonpolar compounds that do not have polar
groups and are not water soluble, or materials
on which surface the water does not adhere are
commonly indicated as hydrophobic. Both the
polar and the hydrophobic character of a com-
pound are reasonably described by the partition
constant (also indicated as partition coefficient)
between octanol and water Kow (or Pow). The
experimental values for Kow are known for
FIGURE 1.1.7 Schematic description of size exclusion many compounds, and for their evaluation there
process. are available several computer programs (e.g.,

MarvinSketch 5.4.0.1, ChemAxon Ltd., [8], EPI publications such as peer reviewed papers,
Suite [9]), as well as extensive tables [10,11]). books (see e.g., Refs. [12e14]), and information
Positive values for log Kow indicate a hydropho- on the web. The main application of analytical
bic character for the compound X, larger values HPLC is in quantitative analysis. However, qual-
indicating higher hydrophobicity. Molecules itative analysis by HPLC is also common,
with low or negative values for Kow are although it has some limitations. Analytical
frequently indicated as polar, although there is HPLC is also used for other purposes than
not a direct relation between Kow and the charge “analytical,” such as the determination of certain
distribution in the molecule. physical parameters of compounds (e.g., octa-
In the body of this book, the polarity will be nol/water partition coefficient), and for other
further discussed and clarified. However, the applications.
concept of polar and nonpolar (hydrophobic) Qualitative analysis is based in HPLC on two
compounds or materials will be frequently different types of parameters, one type being
used in the “imprecise” manner. For mobile related to the separation process and the other
phases, the extension of the concept of polarity related to the detectors utilized. The main
is immediate, being based on the polarity of the parameter from the separation process used in
molecules of the phase. For stationary phases, qualitative analysis is the retention time tR which
the polarity refers to the nature of the stationary is specific for the analytes in a specific separa-
phase surface or of its active part. tion. The expected tR for an analyte in a given
As a conclusion, HPLC techniques can be separation can be determined using a standard,
differentiated based on a number of criteria. and the tR can be used to assess the presence or
Each selected criterion has advantages and dis- absence of the analyte peak in the chromatogram
advantages. For example, the type of interac- of a sample. This procedure, although very use-
tions for a particular separation is not always ful in many applications, has several shortcom-
well understood, and the “polar” or “nonpolar” ings, including the need to use standards in
nature of the interactions involved in the sepa- order to know at which tR the analyte of interest
ration is sometimes difficult to quantify. Also, is eluting and as a result the inability to identify
the physical criteria such as particle dimension an unknown compound, and the possibility that
in the chromatographic column or the scale of other compound may elute at the same tR as the
the HPLC equipment are available in a range analyte which would lead to interferences and
and the limits used for the classification are sub- incorrect identifications. These make the use of
jective. For this reason, the HPLC classifications tR a relatively weak procedure for qualitative
should be viewed mainly as an attempt to have identification.
models, and sometimes a particular HPLC type When the parameters related to the separation
can be classified in more than one way. process are not sufficient (or fail) to provide
qualitative identification of a compound, the in-
formation from the detection may provide the
solution. A variety of detectors are used in
Applications of HPLC in chemical
HPLC, some being indicated as “universal”
analysis meaning they respond to all analytes but do
The utility of HPLC is very diverse, including not provide any qualitative information (such
the utilization for analytical purpose, but also for as refractive index detectors). Other detectors
preparative purposes. The subject of preparative provide only partial information that is not in it-
HPLC is beyond the purpose of this book and it self sufficient for positive identification of an an-
is reported in the literature in a variety of alyte (e.g., UV adsorption). Detectors such as

mass spectrometers (MS) or MS/MS offer more other procedures. Depending on the detection
detailed insight regarding qualitative peak technique and the analyte properties, some
identification. However, the lack of fragmenta- HPLC analyses can provide results even for ul-
tion of the analyte in LC/MS and the depen- tralow traces of a compound (below ng/mL
dence on operational conditions of the mass level). The versatility and high sensitivity of
spectra obtained using LC/MS/MS instrumen- HPLC has contributed to its success and wide-
tation make the process of compound identifica- spread use. An exceptionally large number of
tion still relatively difficult in HPLC. Progress in methods using HPLC quantitation procedures
MS identification of unknown compounds has have been published. Further discussion on
been done, for example, by using very high quantitative procedures used in HPLC is given
accuracy in mass measurement for the parent in Section 3.4 and 18.2.
ion of the analyte and for its fragments (e.g., us-
ing Orbitrap or cyclotron technologies). Also,
specific computer programs (Mass Frontier,
Nonanalytical applications of analytical
SmileMS) provide help for the identification of
unknown compounds. However, in many
HPLC
cases, the compound identification capability, HPLC with various detection techniques is
even using MS or MS/MS detection, is not not only an analytical tool for the determination
used for the discovery of the composition of of chemical species in various matrices, but also
an unknown compound, but for the positive it can be useful in estimating physical parame-
identification of a known analyte, corroborated ters that are otherwise difficult to be measured
with the retention time of its standard, previ- by other approaches [15]. For example, HPLC
ously analyzed. The confirmation of the peak can be used for the evaluation of molecular de-
for a specific analyte in a chromatogram using scriptors such as acidity/basicity constant, hy-
MS detection (typically based on molecular drophobicity, hydrophilicity, dipole moment,
ion and two or three confirmation ions) is an molecular polarizability, and other parameters
important and common procedure in HPLC [16]. Solubility data in various solvents, thermo-
practice. The use of standards with labeled dynamic values for the interphase partition,
isotopes for the analytes (e.g., deuterated ana- molecular interactions, or structural molecular
lyte) spiked in the sample is also a common features [17] are only a few aspects that can be
practice for peak identification in LC/MS and studied using HPLC, under various separation
LC/MS/MS. Although the retention time of types [18]. Quantitative relationships between
the isotope labeled standards may vary slightly molecular descriptors and HPLC retention are
from that of the analyte itself, peak identifica- often used in the literature for the characteriza-
tion is significantly facilitated using this tion of the separation and to predict the chro-
technique. matographic behavior of other compounds
Quantitative analysis is the main use of [19]. Due to the similarities between some natu-
HPLC. Once separated, the concentration of ral systems and some HPLC partition processes,
the analytes in the sample or their amount in the term of biomimetic liquid chromatography
the injected sample can be obtained from the has been suggested in the literature for complex
chromatographic peak area (or height). Peak types of distribution of organic species between
areas (or peak heights) in the chromatogram mobile phase and stationary phase with multi-
are dependent on the concentration or the ple functionalities [20e23]. Biomimetic HPLC is
amount of analyte, and quantitation is done us- considered to mimic the lipid environment of
ing calibration curves with standards, or by a membrane and they can be used in the

1.2 Main types of HPLC 11
assessment of interactions between molecules phase is known as isocratic HPLC, while that per-
and biological membranes or for the estimation formed with a mobile phase that changes contin-
of permeability through cell membranes [24]. uously in composition during the separation
according to a linear or nonlinear elution pro-
gram is known as gradient HPLC (a step change
in mobile phase composition can also be used).
1.2 Main types of HPLC
Gradient HPLC allows a change in the polarity
and/or in the pH of the mobile phase during
Criteria for the classification of HPLC
the separation and significantly increases the
procedures versatility of HPLC. When a sample contains sol-
HPLC is comprised of several similar tech- utes with very different properties and when a
niques having in common the use of a liquid constant composition of the mobile phase (iso-
mobile phase passing a stationary phase with cratic conditions) is used, the solutes may leave
the result of a “high performance” separation the chromatographic column at very different
of a mixture of compounds. Various versions of times. This may be seen as an advantage for
HPLC have specific differences and also the separation, but when the retention time of
different applications. For this reason, the some of the solutes become unacceptably
HPLC techniques are classified in various types, long, the change in the solvent composition (by
the classification being done based on a number using gradients) is necessary to speed up the
of criteria, such as the separation principle (in separation.
some separations more than one principle of sep- The differences in the size of the particles used
aration may have a contribution) or the scale of in the chromatographic column offer one more
the utilization. Additional differences have criterion for HPLC classification. The size of the
been used to distinguish more types of HPLC. particles that fills the chromatographic column
This may include the nature of the stationary affects the peak width and therefore the separa-
phase and mobile phase and/or the type of inter- tion. In common HPLC, the size of the particles
actions that describe the energetics of the pro- in the chromatographic column is indicated by
cess. It should be noted that the classification an average diameter, different columns having
based on types of molecular interactions indi- the particles of values between 1.7 and 10 mm.
rectly includes differences in the nature of sta- The HPLC techniques that use in the chromato-
tionary and mobile phase. For example, ion graphic column (or cartridge) very small parti-
exchange chromatography is practiced specif- cles, (e.g., with a diameter below 2.5 mm), are
ically on an ion exchange stationary phase and usually indicated as Ultra Performance Liquid
not on a reversed phase or a bioaffinity one. Chromatography or UPLC. The very small parti-
However, the type of molecular interaction alone cles require additional modifications in the
was not viewed as sufficient for the differentia- UPLC technique, such as significantly higher
tion of some types of HPLC. Based on the nature operation pressure for the mobile phase. The
of the stationary phase and mobile phase, several use of small particles in the columns can lead
types of HPLC can be differentiated. not only to better separations, but also to faster
Other HPLC characteristics are also used for ones, and is one of the modern developments
differentiating the HPLC types. One such charac- of HPLC. Another development in HPLC is the
teristic is related to the composition of the mobile use of monolithic columns made of a single piece
phase, which can be kept constant during the of a solid porous material.
separation or can be modified. The HPLC per- Temperature can be another criterion to
formed at a constant composition of the mobile differentiate HPLC processes. Based on this

parameter, the HPLC types are classified as (1) with bigger particles compared to analytical
Low temperature (below freezing point of water, HPLC [30].
down to 10 C), (2) Usual range temperature
(20e60 C), and (3) High temperature (up to
250 C). Most separations are performed at usual A classification of HPLC types based on
temperatures. Low temperature techniques are the nature of stationary and mobile phase
applied especially for certain chiral separations.
High-temperature separations can be used for a A variety of HPLC types were differentiated
number of applications taking advantage of the in the literature, some of these types being rather
modification of solvents properties as tempera- similar and others having significant differences.
ture increases. Water, for example, shows a The differentiation was based on various criteria
decrease in polarity at temperatures between such as the nature of the stationary phase, the na-
100 and 250 C and can be used as solvent in ture of the mobile phase, the type of interactions
RP-HPLC. This particular mode of separation assumed to lead to the separation, but also the
has been denoted as superheated water, pressur- range of concentration of specific solvents in
ized water, or subcritical water chromatography the mobile phase (e.g., of water), etc. Multiple
(as the temperatures used are lower than the crit- types of interactions are usually involved in
ical temperature of water at 374 C) [25]. The use HPLC separations, but in many cases it is
of water as a mobile phase can provide an envi- possible to indicate the dominant one. A com-
ronmentally friendly “green” method of chro- mon classification of the main types of HPLC is
matographic analysis. given in this section. Because different HPLC
A different classification, important for prac- types have different characteristics and different
tical purposes, is based on the scale of the applications, it is important to understand their
HPLC equipment. Three general types of chro- differences and select the appropriate HPLC
matography can be distinguished in this way: type for solving a specific separation/analysis
(1) Analytical HPLC, (2) Semipreparative HPLC, problem.
and (3) Preparative (Large scale) HPLC. Each of (1) Reversed-phase HPLC (or RP-HPLC) is the
these types covers in fact a range of dimensions most common HPLC technique, and a very large
[3]. For example, analytical HPLC can be further number of compounds can be separated by RP-
differentiated based on the dimensions of the HPLC. This type of chromatography is
HPLC column in the following subtypes: con- performed on a nonpolar stationary phase with
ventional, narrowbore, microbore, micro-LC- a polar mobile phase containing water. A wide
capillary, and nano-LC-capillary [26e28]. In variety of nonpolar stationary phases is
this book, most discussions will refer to conven- available, and RP-HPLC is very likely the most
tional, narrowbore, and microbore analytical common type of chromatography used in
HPLC. Analytical HPLC performed using micro- practice. The stationary phase for RP-HPLC can
capillary and nanoscale columns (still filled with be obtained, for example, by chemically bonding
a bed of particles) are less used in practice and long hydrocarbon chains on a solid surface such
require specialized equipment [29]. In case of as silica. The most common chain bound to silica
preparative HPLC, the primary goal of separa- is C18 (it contains 18 carbon atoms), which has a
tion is to collect fraction of pure compounds high hydrophobic character. The bonded phase
with high yields. This technique uses larger chro- hydrophobicity may vary depending on the
matographic columns and stationary phases nature of the substituent. For example, C18

bonded phase has a higher hydrophobicity than This type of chromatography is very similar to
C8 bonded phases. Polymeric materials are also RP-HPLC, with the difference of having a
used as RP-HPLC stationary phase. The mobile special mobile phase (ion pair RP). In the
phase in RP-HPLC is typically a mixture of an mobile phase of ion-pair chromatography, a
organic solvent (CH3CN, CH3OH, isopropanol, reagent is added, which interacts with the ions
etc.) and water, with a range of content in the of the analytes and forms less polar compounds
organic solvent. Small amounts of buffers can that can be separated based on hydrophobic
also be added to the mobile phase in RP-HPLC. interactions with the stationary phase. For
The main equilibrium in RP-HPLC is example, acids that are polar and have small
partition, and the main type of molecular hydrophobic moieties can be coupled with
interactions are indicated as hydrophobic basic reagents with a large hydrophobic moiety
interactions. These hydrophobic interactions that produces “ion pairs” amenable to be
(sometimes called hydrophobic forces) are separated by RP-HPLC.
caused by the energies resulting from the
(3) Hydrophobic interaction chromatography (HIC)
reduction of the disturbance of the structure of
is a type of RP-HPLC, sometimes indicated as a
water/polar solvents caused by compounds
milder RP-HPLC, applied to the separations of
with nonpolar moieties. The new hydrogen
proteins and other biopolymers. The technique
bonds and polar interactions formed in the
is based on interactions between nonpolar
water/polar solvents when nonpolar molecules
moieties of a protein with solvent-accessible
are eliminated are a source of energy. The so-
nonpolar groups (hydrophobic patches) on the
called “solvophobic effect” is produced by the
surface of a hydrophilic stationary phase (e.g.,
energy of “cavity reduction around the analyte”
hydrophobic ligands coupled on cross-linked
in water/polar solvent when the analyte leaves
agarose). The promotion of the hydrophobic
the mobile phase and is placed in the nonpolar
effect by addition of salts (such as ammonium
stationary phase. However, depending on the
sulfate) in the mobile phase drives the
structure of the molecule of the analyte, other
adsorption of hydrophobic areas from
types of interactions including polar interactions
the protein to the hydrophobic areas on the
and even ionic interactions may play some role
stationary phase. The reduction of the salting
in RP-HPLC separations. In RP-HPLC, the
out effect by decreasing the concentration of
separation is typically considered to be based on
salts in solution leads to the desorption of the
the partition of the analyte between the
protein from the solid support.
stationary phase (viewed as an immobilized
hydrophobic liquid) and the mobile phase. (4) Nonaqueous reversed-phase chromatography
Nevertheless, some experimental results in RP- (NARP) is an RP-HPLC type utilized for the
HPLC can also be explained by adsorption separation of very hydrophobic molecules such
equilibrium. The exceptional utility of RP-HPLC as triglycerides. In this type of chromatography,
is based on the fact that most organic the stationary phase is nonpolar (similar to RP),
compounds have at least some hydrophobic while the mobile phase, although less nonpolar
moiety in their structure, and this technique can than the stationary phase, is nonaqueous
be used for their separation. (usually a mixture of less polar and more polar
organic solvents) and capable of dissolving the
(2) Ion-pair chromatography (IPC) is applied in
strongly hydrophobic molecules.
particular to ionic or strongly polar compounds.

(5) Hydrophilic interaction liquid chromatography this type of chromatography, the ionic stationary
(HILIC) is a type of HPLC applied for polar, phase repels the similar ionic groups of the
weakly acidic, or basic compounds. In this type analyte and allows HILIC type interactions with
of HPLC, the stationary phase is polar and the the neutral polar molecules of the analyte.
mobile phase is less polar than the stationary
(6) Normal phase chromatography (NPC or NP-
phase. HILIC is the “reverse” of RP-HPLC. For
HPLC) is a chromatographic type that uses a
HILIC, the polar stationary phase is typically
polar stationary phase and a nonpolar mobile
made by chemically bonding on a solid support
phase for the separation of polar compounds.
molecular fragments with a polar end group
The nonpolar mobile phases used in this type of
(diol, amino, special zwitterionic, etc.). The
chromatography are solvents such as hexane,
chromatography performed on bare silica
CH2Cl2, tetrahydrofuran, etc., that are not water
supported with free silanol (/SieOH) groups
soluble. In normal phase chromatography, the
can also be considered as HILIC, depending on
most nonpolar compounds elute first and the
the mobile phase. The mobile phase in HILIC is
most polar compounds elute last. Normal phase
less polar than the stationary phase and contains
chromatography does not have a major
water soluble solvent such as CH3OH or
difference from HILIC except the absence of
CH3CN, and also a certain proportion of water.
water in the mobile phase. Because NPC was
The separation is based on the difference in
identified as a separate type for much longer
polarity between the molecules. Ionepolar
time than HILIC, it is common in the literature to
interactions and even hydrophobic interactions
identify HILIC as a subtype of normal phase
may also play a role in separation. The type
chromatography, and not the other way around.
adsorption of partition for HILIC is difficult to
A polar organic normal phase is sometimes
assess. Viewed as having the separation
mentioned as a type of chromatography when
equilibrium based on the interaction of a solid
the nonaqueous solvent contains polar additives
surface with the molecules from a liquid, HILIC
such as trifluoroacetic acid.
can be considered adsorption chromatography.
However, a (polar) bonded phase may be seen (7) Aqueous normal phase chromatography (ANPC
as a stationary liquid phase, and in this case, or ANP) [31,32] is a technique performed on a
HILIC is a type of partition chromatography. special stationary phase (silica hydride) and the
When the separation is done on zwitterionic mobile phase covers the range including the
phases, HILIC chromatography is sometimes types used in reversed-phase chromatography
indicated as ZIC. and those used in normal phase
HILIC separations can also be performed on chromatography. The mobile phases for ANP
an ion exchange stationary phase with the are based on an organic solvent (such as
mobile phase containing a high proportion of an methanol or acetonitrile) with a certain amount
organic solvent. This type of separation is of water such that the mobile phase can be both
sometimes indicated as eHILIC or ERLIC (from “aqueous” (water is present) and “normal” (less
electrostatic repulsion hydrophilic interaction polar than the stationary phase). Polar solutes
chromatography). This technique can be cationic are most strongly retained in ANP, with
eHILIC or anionic eHILIC, depending on the retention decreasing as the amount of water in
nature of the ion exchange stationary phase. In the mobile phase increases.

(8) Cation exchange chromatography is a type of exchange chromatography usually consists of
HPLC used for the separation of cations buffer solutions.
(inorganic or organic). In this HPLC type, the
(10) Ion exchange on amphoteric or on zwitterionic
retention is based on the attraction between ions
phases is a type of IEC very similar in principle
in a solution and the opposite charged sites
with the cation exchange or anion exchange IEC.
bound to the stationary phase. In ion exchange
The stationary phase for this type of IEC
chromatography (IEC or IC), the ionic species
contains groups that have an amphoteric
are retained on the column based on coulombic
character or in the case of zwitterionic phases,
interactions. In cation exchange
both anionic and cationic groups. The mobile
chromatography, the ionic compound consisting
phase in these types of chromatography also
of the cationic species Mþ in solution is retained
consists of buffer solutions.
by ionic groups covalently bonded to a
stationary support of the type ReX. The ion (11) Ion exclusion chromatography is an HPLC
exchange material (e.g., an organic polymer with technique in which an ion exchange resin is used
ionic groups) is not electrically charged, and for the separation of neutral species between
therefore the initial form of the cation exchange them and from ionic species. In this technique,
already has an ionically retained cation in the ionic compounds from the solution are rejected
form ReXCþ. The separation is achieved when by the selected resin (through the so called
different molecules in solution have different Donnan effect), and they are eluted as
acidic or basic strength. For example, for a cation nonretained compounds. Nonionic or weakly
exchange material, one species (e.g., Cþ) that is ionic compounds penetrate the pores of the resin
bound to the ReX substrate is replaced by a and are retained selectively as they partition
stronger cationic species (e.g., M þ) such that Mþ between the liquid inside the resin and the
is retained from the solution, while Cþ passes mobile phase (Donnan effect or GibbseDonnan
into the mobile phase. Two different cations effect describes the distribution of ions in
from solution, Mþ þ
1 and M2 , can be separated solution in two compartments separated by a
based on their retention strength. semipermeable membrane).
(9) Anion exchange chromatography is a type of (12) Ligand exchange chromatography is a type of
HPLC used for the separation of anions chromatography in which the stationary phase
(inorganic or organic). This HPLC is similar in is a cation exchange resin loaded with a metal
principle with cation exchange type, but the ion (e.g., of a transitional metal) that is able to
anionic species Be from solution are retained by form coordinative bonds with the molecules
covalently bonded ionic groups of the type ReY from the mobile phase. The elution is done with
þ
. Similarly to cation exchange stationary a mobile phase able to displace the analyte from
phases, an anion exchange is initially in the form the bond with the metal, and the separation is
ReYþA. For an anion exchange material, the based on the differences in the strength of the
anion A previously bound is replaced on the interaction (of coordinative type) of these solutes
resin by the anion B, and two different anions with the bonded metal ion.
B
1 and B2 are separated based on their different
(13) Immobilized metal affinity chromatography is
retention strengths. The mobile phase in ion
closely related to ligand exchange

chromatography and uses a resin containing macromolecules and of macromolecules from

chelating groups that can form complexes with small molecules. GFC is sometimes indicated as
metals such as Cu2þ, Ni2þ, Zn2þ, etc. The metal aqueous SEC.
ions loaded on the resin still have coordinative
(16) Gel permeation chromatography (GPC) is
capability for other electron donor molecules
another type of SEC, the only difference from gel
such as proteins. The retained analytes can be
filtration being the mobile phase, which in this
eluted by destabilizing the complex with the
case is an organic solvent. The technique is used
metal, e.g., by pH changes or addition of a
mainly for the separation of hydrophobic
displacing agent such ammonia in the mobile
macromolecules (such as solutions of certain
phase.
synthetic polymers). GPC is sometimes
(14) Ion-moderated chromatography is an HPLC indicated as nonaqueous SEC.
technique similar to ligand exchange
(17) Displacement chromatography is a
chromatography with the difference that the
chromatographic technique where all the
stationary phase loaded with the metal ion (e.g.,
molecules of a sample are initially retained on
Ca2þ, Naþ, Kþ, Agþ, or even Hþ) does not form
a chromatographic column (loading phase).
coordinative bonds with the analyte, the
After the sample is loaded, a “displacement”
interactions being based mainly on polarity.
reagent dissolved in the mobile phase is passed
(15) Gel filtration chromatography (GFC) is a type through the column and elutes the specific
of size exclusion chromatography (SEC) in retained molecule. The method is more
which the molecules are separated based on frequently applied as a preparative
their size (more correctly, their hydrodynamic chromatographic technique than as an HPLC
volume). In gel filtration, an aqueous (mostly analytical method.
aqueous) solution is used to transport the
(18) Affinity chromatography is a liquid
sample through the column and is applied to
chromatographic technique typically used for
molecules that are soluble in water and polar
protein and other biomolecules separation when
solvents. Size exclusion chromatography uses
commonly indicated as bioaffinity
porous particles with a variety of pore sizes to
chromatography. It can be practiced on a variety
separate molecules. Molecules that are smaller
of specifically made stationary phases that allow
than the pore size of the stationary phase enter
selective retention of the analytes based on
the porous particles during the separation and
affinity interactions.
flow through the intricate channels of the
stationary phase. Small molecules have a long (19) Chiral chromatography on chiral stationary
path through the column and therefore a long phases is a type of HPLC used to separate chiral
transit time. Some very large molecules cannot compounds. Specific applications require the
enter the pores at all and elute without retention separation of chiral compounds, although
(total exclusion). Molecules of medium size enter regular chromatography is much more common
only some larger pores and not the small ones, than chiral chromatography. Chiral
and are only partly retained, eluting faster than chromatography still has numerous
small molecules and slower than the very large applications, particularly in the analysis of
ones. The separation of small molecules between pharmaceutical compounds. The technique
themselves is not typically achieved, and the typically requires chiral stationary phases
technique is utilized mainly for the separation of containing chiral selector groups.

1.3 Flow of a typical HPLC analysis 17
(20) Chiral chromatography on achiral stationary TABLE 1.2.1 Separation principle and main types of
phases is also possible for some chiral solutes, by HPLC.
using chiral modifiers in the mobile phase, Separation
although the stationary phase is not chiral. This interactions Types of HPLC
type of HPLC separates the diastereoisomers
Hydrophobic (1) Reversed phase (RP)
formed between the analytes and the chiral (2) Ion pair
interactions
modifier from the mobile phase. (3) HIC
(4) Nonaqueous reversed phase
(21) Multimode HPLC is a type of (NARP)
chromatography in which the stationary phase
Difference in (5) HILIC
contains by purpose more than one type of polarity (6) Normal phase (NPC)
functionality, for example, some with bonded (7) Aqueous normal phase (ANP)
nonpolar groups (e.g., C18), and some with ionic Ion interaction (8) Cation exchange
groups (e.g., SO-3). This type of stationary phases (9) Anion exchange
is able to participate to multiple interactions (10) Ion exchange on amphoteric and
with solutes having different molecular zwitterionic phases
(11) Ion exclusion
characteristics, thus contributing to their (12) Ligand exchange
retention on the stationary phase [33]. This type (13) Immobilized metal affinity
of character can also be encountered (14) Ion moderated
unintentionally on columns made using as a Size exclusion (15) Gel filtration
stationary phase a silica support covered with (16) Gel permeation
silanol groups, and also with hydrophobic Displacement (17) Displacement
groups (such as C18). In most cases, the presence
Bioaffinity (18) Bioaffinity (not always HPLC)
of two main types of interactions (e.g., polar and
hydrophobic) is not desirable, but in some Chiral (19) Chiral stationary phase
(20) Chiral mobile phase
instances dual properties of a stationary phase
can be used to the advantage of the separation. Various principles (21) Multimode
together
Relation between the type of HPLC,

equilibrium type, and molecular separation interactions can be noticed. The rela-
interactions tion between the HPLC main groups and the
The identification of equilibrium type (e.g., equilibrium type or molecular interaction types
partition, adsorption, ionic, size exclusion) for should not be viewed as rigid. The main separa-
each type of HPLC is not a straightforward sub- tion principles and the corresponding HPLC
ject [34]. It is possible that more than one such types are summarized in Table 1.2.1.
equilibrium takes place in a specific HPLC
type, and in some cases it is difficult to decide
1.3 Flow of a typical HPLC analysis
based on experimental data which equilibrium
type is involved in the separation. Also, more
than one type of interactions is present in most
General aspects
HPLC separations. However, an association be- The practical steps for an HPLC analysis are
tween different types of HPLC and different conducted based on a number of decisions

regarding sample collection (procedure, quan- molecules based on their molecular weight (in
tity, number of replicates), sample preparation fact hydrodynamic volume) are performed by
(choice of cleanup, concentration, and/or deriv- size exclusion. Bioaffinity chromatography is
atization), type of HPLC as well as type of widely utilized for the separation of biological
required type of information (qualitative or macromolecules. Details regarding selection of
quantitative measurements). The analytical chro- a type of HPLC are given in Section 18.1.
matography can be considered the core of the The purpose of analysis is another deter-
process, and it includes the identification and mining factor in the selection of HPLC type. In
measurement of the analytes. The choice of this choice, it is important to know if the analysis
HPLC as the analytical step is done for is performed for the separation by molecular
numerous types of samples. This includes small weight, for specific identification and quantita-
molecules with medium and low volatility, as tion of components, or for separation and quan-
well as larger molecules including a wide range titation of enantiomers. Other factors also
of synthetic and biopolymers. HPLC has the influence the choice of HPLC, such as availabil-
capability of separating complex mixtures and ity of equipment, requirements regarding anal-
performs accurate quantitation with extreme ysis time, the number of samples to be
sensitivity. analyzed, availability of specific materials
required for the analysis (columns, solvents,
etc.), restrictions regarding safety (e.g., the na-
ture and volume of solvents to be disposed),
Selection of the type of HPLC for a
level of training of the operator, etc. In this sec-
particular application tion, only a general guidance regarding the selec-
An important part of the information step in tion of the HPLC type is provided, and this
chromatographic analysis is the choice of the selection is based solely on the nature of sample.
type of HPLC that should be used. This is done The selection of a particular type of chroma-
based on the nature of sample, instruments tography for a specific analysis is a complex pro-
availability, as well as other factors such as cost cess, the previous discussion being only a
and time of analysis. Once the HPLC technique schematic guide which is far from being compre-
is selected as the core analytical procedure, hensive. Numerous sample/analyte details may
further decisions should be made regarding the determine the final choice of a specific chromato-
type of HPLC. The selection of an HPLC type graphic separation. This book discusses various
for the analysis of a particular set of samples is aspects of separation in analytical HPLC and
not always simple. However, there are some provides guidance regarding choices of type of
general rules that may be used as guidance. HPLC, specific columns, and mobile phases to
This choice is determined primarily by the na- be used for a successful analysis.
ture of the sample with its analytes and matrix.
Reversed-phase chromatography, for example,
is commonly used for a wide range of com-
Sample collection and sample preparation
pounds, including various organic molecules
for HPLC
that have some hydrophobic moiety. More polar
molecules are typically analyzed using HILIC, Sample collection is a very important step for
ion pair chromatography or in some instances the success of any chemical analysis. The subject
even RP-HPLC can still be used for the separa- is discussed in various books and papers; how-
tion. Ions (inorganic or small organic) are typi- ever, this subject being outside of the purpose
cally analyzed by IC. The separations of large of this book, the reader should refer to the

References 19
Sample collection Sample preparation HPLC
Grinding, sieving
Sample size reduction
Weighing Analyze
Obtain raw
Dissolution (physical, processed
sample
chemical) or extraction sample
from matrix
Sample cleanup
Fractionation (if needed)
Concentration
Derivatization
FIGURE 1.3.1 Diagram indicating the place of a sample preparation in chromatographic analysis involving dissolution or
extraction from matrix, cleanup, fractionation, concentration, and derivatization.
dedicated literature (e.g., Refs. [1,35]). After sam- The process of sample preparation and its place
ple collection, the analysis proceeds with the in chromatographic analysis is schematically
sample preparation, in accord with the selected shown in Fig. 1.3.1.
analysis type. Again, numerous procedures are Sample preparation is usually described for
described in the literature for sample prepara- each HPLC analytical procedure when applied
tion [1]. Sample preparation may target the ma- for a practical analysis and covers a large part
trix of the sample, the analytes, or both. One of published literature on HPLC. Several books
common operation in sample preparation is the describing the general principles and various as-
dissolution of the sample if the sample is solid. pects of sample preparation for chromatography
Then, the matrix is usually modified during a also have been published (e.g., Refs. [36,37]).
cleanup, fractionation, and concentration of the
sample. The proper processing of the sample References
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(2013) 211e226. actions for HPLC, J. Wiley & Sons, New York, 1998.
[24] V. David, N. Grinberg, S.C. Moldoveanu, Long range [37] J. Pawliszyn (Ed.), Sampling and Sample Preparation
molecular interactions involved in the retention in Field and Laboratory, Elsevier, Amsterdam, 2002.

C H A P T E R
2
Overview of HPLC instrumentation
and its use
2.1 Description of main components of of HPLC instrumentation is not the main goal
HPLC instrumentation of this book, and more information on the subject
can be obtained from various sources such as
General comments instrument manuals, dedicated books (e.g.,
Refs. [1e8]), peer reviewed papers, or from in-
The HPLC analytical instrument has the role
formation from the internet.
to physically separate totally or partially the
components of a sample in the form of a solution,
and generate an electrical signal recorded as a
Description of a typical HPLC instrument
chromatogram for the separated components. The basic configuration of an HPLC instru-
For this purpose, the instruments allow the injec- ment includes the following modules: (1) a sol-
tion of a measured small volume of sample in a vent supply system (solvent containers and
liquid mobile phase. This mobile phase flows degasser for generating the mobile phase), (2) a
through a chromatographic column where the high pressure pumping system, (3) an injector,
separation takes place, and further through a de- frequently included in an autosampler, (4) a ther-
tector (or detectors) capable of generating an mostatted column holder, (5) a chromatographic
electrical signal. The signal is most frequently column (possibly with a guard column or precol-
used for quantitative measurements of analytes, umn), (6) one or more detectors, and (7) software
although special detectors may provide also for the control of instrument parameters, data
qualitative information. Modern HPLC systems acquisition, and processing. Depending on the
are controlled using complex computer soft- purpose of HPLC, other instrument capabilities
ware. This process can be achieved using a large can be included, such as a cooling system for
number of models of HPLC systems. The con- the autosampler/injector, a fraction collector,
struction of an HPLC instrument may vary column switching for back-flow or multi column
from one manufacturer to another and depends use, etc. The diagram of a simple type of con-
on the intended function and size of the HPLC struction for an HPLC is shown in Fig. 2.1.1 [8].
process. Also, modern HPLC instrumentation Some details on each of the components of an
can be rather sophisticated, and the instruments HPLC system are further discussed in this
are in continuous development. The description section.

https://doi.org/10.1016/B978-0-323-91177-1.00015-6 21 © 2022 Elsevier Inc. All rights reserved.
22 2. Overview of HPLC instrumentation and its use
FIGURE 2.1.1 Schematic diagram of a simple configuration for an HPLC system.
Solvent supply system coming from the solvent reservoir cannot be

eliminated by the degasser apparatus, and these
The role of solvent supply module is to pro- bubbles make their way into the pumps affecting
vide the mobile phase necessary for the HPLC their function. The degasser can be a separate unit
separation. Usually, the solvent is contained in or can be imbedded in the pumping system.
(inert) glass bottles and a first step in using this The degassing is necessary because even very
solvent is the degassing. The degassing can be pure solvents may have dissolved in them small
performed by two procedures, a preliminary quantities of oxygen. This oxygen may be
degassing and an in-line degassing. The prelim- released in the form of very small bubbles in
inary degassing is optional and is done before the HPLC system when a drop in pressure oc-
the mobile phase inclusion in the HPLC flow. curs (e.g., between the chromatographic column
The preliminary degassing is done by sparging and the detector), or when a solvent with high
the solvents with an inert gas (N2, He) at a low solubility for oxygen (e.g., water) is mixed with
flow rate, or by ultrasonication, when the con- another solvent with low solubility for this gas.
tent of the dissolved O2 in solvents is reduced. When this mixing is done before the high pres-
The second type of degassing in an HPLC sys- sure pump, the gas bubbles may lead to pressure
tem is performed in an in-line degasser device. fluctuations of the liquid delivered by the pump.
The principle of this device is to pass the mobile The pressure fluctuation should be low and in
phase through a piece of special polymeric tubing modern HPLC systems it is typically below
placed in a vacuum chamber. The tubing material 0.1% of the nominal pressure. When the gasses
(membrane) has selective permeability to gasses, are not removed from the solvents, even if the
and the (mild) vacuum created by a small pump re- pumps are working properly, pressure fluctua-
duces the content of the gasses from the solvent tions of 4%e6% of the nominal pressure can be
(e.g., Ref. [9]). The degassers, although popular noticed. Dissolved gasses in the mobile phase
in HPLC equipment, also may pose problems in may also influence the injection volume when
specific applications. The polymeric tubing may small sample volumes (e.g., 1e2 mL) are injected.
absorb selectively specific components from the Also, the reading of the detectors can be
solvents and may be a source of contamination perturbed by dissolved gasses. For example, ox-
when changing from one solvent to another. ygen may affect the reading of electrochemical
Also, it should be noted that large bubbles detectors, the fluorescence intensity of certain

2.1 Description of main components of HPLC instrumentation 23
compounds, and the UV absorption at very low filter selected for the filtration must be inert to
wavelength range. the solvent. In the case of solvent mixtures contain-
During degassing, in particular for premixed ing a buffer, the general rule indicates that the
solvents, attention must be paid to avoid buffer solution is made in water, then preferably
changes of solvent composition due to preferen- filtered, and only after that mixed with the organic
tial evaporation of the volatile component. For solvent (assuming correct concentrations and no
example, when ammonia or volatile acids are precipitation after the organic solvent addition,
used as additive in mobile phase to adjust the due to lower solubility of the buffer in the mixture
pH or to facilitate a specific detection, sparging of solvents). The tubing transferring the liquid to
is not recommended since drastic changes in the pump(s) typically has a frit at its mouth.
the pH may occur in time by the volatilization A special type of solvent delivery system can
of these components from the solution. Even be used in ion chromatography. This system is
with common solvent mixtures, some composi- known as eluent generator (Thermo Scientific/
tion changes may occur during sparging. For Dionex).
example, degassing with He for premixed mo-
bile phase consisting in 50% water and 50%
organic solvent (v/v) leads to a loss of 0.005%
for methanol, 0.006% for acetonitrile, or 0.05%
Pumping systems
for tetrahydrofuran [10]. The main pumping system consists of
The transfer of the solvent(s) from the solvent pump(s) able to deliver a constant flow of sol-
containers (bottles) to the pumping system is vent through the injector, chromatographic col-
done through low pressure tubing. The tubes umn, and through the detector(s). The pumps
used for passing the mobile phase through must be able to generate a high pressure, which
the system need to fulfill mainly the requirement is needed mainly to overcome the resistance to
of being inert to the utilized solvents and flow of the chromatographic column. This flow
to stand low pressures up to about 50 psi is characterized by the volumetric flow rate U. In
(1 psi ¼ 6.89476 kPa ¼ 6.89476 102 bar ¼ 6.80460 conventional HPLC systems, the pumps are usu-
102 atm; 1 bar ¼ 14.5037738 psi). Fluorocarbon ally capable of delivering U between 0.1 mL/
polymers such as Teflon are common materials min and 5 mL/min (or 10 mL/min for some in-
used for this type of tubing, but polypropylene is struments) and generate up to 6000 psi (about
also used. The solvent supply system of an 400 bar). New developments in using very fine
HPLC has one or more reservoirs for the solvents particles in the chromatographic column require
used as mobile phase. For HPLC performed in iso- higher pressure and sometimes capability to pro-
cratic conditions and using a pure or a premixed duce flows at less than 0.1 mL/min. These in-
solvent, only one reservoir is necessary. However, struments indicated as UPLC or U-HPLC can
it is common in HPLC to use gradient separations, generate up to 19,000 psi (about 1300 bar) at
or to use an isocratic separation but to generate a 1.0 mL/min [11] (lower maximum pressures
mixture of solvents using the pumps. In this case, are allowed at higher flow rates). For the HPLC
two (or more) solvents that are mixed with the systems used for other purposes than analytical,
pumping system in variable proportions are pumping parameters can vary significantly. The
required. The reservoirs must be clean and inert flow from the pumps (volumetric flow rate U)
to the solvents they contain. The solvents from must be constant, without fluctuations or only
the reservoirs must be free of particles, and they with very small ones. This requirement is neces-
are either purchased as HPLC grade or/and sary mainly for the detectors, where the signal
filtered through 0.45 mm filters before use. The may fluctuate when the flow rate varies.

Most high pressure pumps used in analytical shaped driving cams or of stepper-driven motors
HPLC are reciprocating pumps. A single piston allows the generation of an almost continuous
reciprocating pump consists of a cylinder with flow of liquid. The dual piston pumps may
a reciprocating plunger in it, and two valves be connected in parallel or in series. An
mounted in the head of the cylinder. The liquid accumulator-piston design with the pumps in se-
enters the cylinder through an inlet (suction) ries is shown schematically in Fig. 2.1.3. This
valve and is pushed through a discharge valve. type of system also requires two pistons but
During the suction, the plunger retracts and the only three valves in order to achieve the task of
inlet valve opens causing the admission of fluid generating a continuous flow.
into the cylinder, while the discharge valve is Modern systems are able to deliver flow with
closed. In the forward stroke, the plunger pushes a precision of about 0.07% relative standard de-
the liquid out through the discharge valve, while viation (RSD%) and a flow accuracy of less
the inlet valve is closed. However, the fluid flow than 1% from the nominal value. Because the
from a single piston reciprocating pump (and pumps must deliver flow at high or very high
therefore the pressure in the system) has a pul- pressure, their construction requires special ma-
sating profile. When the piston is moved by a cir- terials such as inert steel body, sapphire or
cular motion of a driving cam, the flow rate has a ceramic pistons, high precision valves that do
half sinusoid shape as shown in Fig. 2.1.2. not have any leaks, special polymeric seals, etc.
This type of flow is not suitable for HPLC. For ion chromatography, the whole pumping
Dual piston pumps consisting of two recipro- system is typically made of strong polymeric
cating pumps that alternate the forward stoke materials such as polyetheretherketone (PEEK).
are able to generate flow with only one zero In addition to the pumps and valves specially
flow point per cycle. However, with this setup, designed, the flow without fluctuations from the
the flow is still fluctuating. The use of specially pumping system can be achieved using a
FIGURE 2.1.2 Flow from a single piston reciprocating pump when the piston is moved by a circular motion of a driving
cam.
FIGURE 2.1.3 Flow from a dual accumulator-piston reciprocating pump when the pistons are moved by stepper-driven
motors that allows low pulsation.

pressure pulsation damper. Pressure dampening solvents are delivered further to one (dual pis-
is done, for example, by passing the fluid ton) high pressure pump. Low pressure mixing
through a cell with a diaphragm wall that com- has the advantage of using a single high pressure
pensates the pressure variation. The pulsation pump (that is typically expensive), and has more
with dampening can be reduced to less than flexibility in choosing a variety of solvents (in
2% variation in pressure. Various models of systems with four proportioning valves). How-
dampers are available, and most of them have ever, the changes in the mobile phase composi-
a volume of around 500 mL, in order to assure a tion when using a low pressure mixing system
small delay volume in the delivered fluid. are taking place more gradually than for the
A typical delay volume for an analytical HPLC other systems (the change in composition is not
system is around 700 mL. The pumping equip- instantaneous). Also, low pressure mixing may
ment of modern HPLC frequently has the capa- be prone to the formation of small bubbles of
bility to detect any leakage, communicating to gas in the mixed solvent, when the solubility of
the controlling computer of the HPLC to stop oxygen, for example, is higher in one solvent
the operation when a leak is detected. than in the mixture. These bubbles may enter
A dual piston pump can handle only one sol- the high pressure pump and generate flow
vent and can be applied for isocratic separations fluctuations.
that use a pure or a premixed solvent. However, In high pressure mixing, two high pressure
since in HPLC it is frequently necessary to use (dual piston) pumps are used, and the ratio of
gradient separation, instruments that handle solvents in the mobile phase is controlled by
more than one solvent were developed. This the flow rate of the high pressure pumps. The
type of instrument is also used to generate a sol- mixing of the two solvents A and B can be
vent mixture of a desired composition, even achieved either in a specially designed mixer
when this composition is not changed during (typically with volume of less than 500 mL) or
the separation (isocratic conditions). There are in a mixing T (with virtually zero volume).
three basic procedures to physically achieve the High pressure mixing is considered to provide
mixing of solvents: (1) low pressure mixing, a more precise control of the composition of
where the solvents necessary for the gradient the mobile phase (with a typical composition
are premixed with a low pressure pump con- precision with less than 0.15% RSD% at 1 mL/
nected in front of the high pressure pump, (2) min flow), and does not have the problem of for-
high pressure mixing that uses usually two mation of bubbles caused by the difference in
high pressure pumps with each one dedicated solubility of oxygen in the mixed solvent
to one solvent and with the mixing the flows in compared to that in one of the components.
a low volume mixer, and (3) hybrid mixing However, the cost of high pressure pumps is a
that uses a high pressure pump with two or three disadvantage for this type of mixing. Most mod-
proportioning inlet valves. ern HPLC systems with high pressure mixing
In low pressure mixing, two or more (usually have two pumps and the capability of switching
four) solvents can be blended at the desired between two solvent pairs (A1, B1 and A2, B2).
composition by using a low pressure pump Since instruments with low pressure mixing
with several proportioning inlet valves and instruments with high pressure mixing are
controlled by a computer. The valves open both common in laboratories, when transferring
repeatedly for a short period of time (typically an established analytical technique from one in-
less than one second), the duration of time the strument to another, attention should be paid
valve is opened being proportional with the to the type of instrument. In both types of chro-
desired mobile phase composition. The mixed matographic systems, there is a specific volume

passing the system from the point at which the During the separation, the composition of the
mobile phase solvents are mixed until they reach mobile phase can be kept constant for some in-
the head of the chromatographic column. This tervals of time and modified for other periods
volume is known as dwell volume VD. The dwell of time. The modern instruments are commonly
volume is typically different in low pressure controlled using a computer with a dedicated
mixing systems (2e4 mL) and in high pressure program that assists in generating a specific
mixing systems (1e3 mL). Special instruments, gradient using a gradient time table. Based
such those used in microscale HPLC, may have on the gradient time table, the computer controls
smaller dwell volumes (less than 300 mL). For the pumping system that physically generates
certain applications using gradient separations the desired mobile phase composition by
on common HPLC systems, differences can be mixing in the correct proportion of the solvents
seen when working with one type of instrument from the solvent supply system. The gradient
or with the other, although the gradient program starts when the sample is injected. After the
is the same. This is in particular caused by the gradient ends, the HPLC chromatograph is
differences in the dwell volume from one system made ready for the next injection. The total run
to another. time of the chromatogram, starting with the
Hybrid mixing uses a system of two recipro- moment of injection until the end of the chro-
cating high pressure pumps with proportioning matographic run, is sometimes referred to as to-
inlet (suction) valves. Low pressure mixing sys- tal cycle time. In a gradient separation, the dwell
tems with four proportioning valves and one volume VD of the system creates a dwell time
dual piston high pressure pump, as well as tD. Because of the dwell time, there is a delay be-
high pressure mixing systems with two high tween the change of composition at the point of
pressure pumps (each one dual piston), are solvent mixing (set in the time table) and the
much more common than hybrid systems. change in composition at the head of chromato-
In order to be able to modify the composition graphic column. Therefore, when attempting to
of the mobile phase in gradient HPLC, the sol- modify the retention time of a peak by using a
vents that are blended in specified proportions “stronger” solvent, this change should be done
should be perfectly miscible. Particular care in the gradient time table ahead of the peak
must be paid to the solubility differences of retention time.
certain additives present in one solvent when The modification in concentration between
another solvent is added. For example, it is com- two changing points of the gradient can be
mon in HPLC to use buffer solutions. These solu- linear. For a gradient starting at time t1 with
tions can be easily made in water by adding the concentration C1 of component A and ending
mixtures of salts and acids or salts and bases. at time t2 with the concentration C2, at an inter-
When a water solution containing these types mediate point at time t the concentration of
of additives is mixed with an organic solvent component A in a binary system can be obtained
(such as CH3OH or CH3CN), the solubility of using the following expression:
the additives in the mixed organic/aqueous so-
ðt t1 Þ
lution is drastically diminished. The formation CðtÞ ¼ C1 þ ðC2 C1 Þ (2.1.1)
of precipitates following the mixing must be ðt2 t1 Þ
carefully avoided and only buffers at low In most HPLC separations, the mobile phase
concentration of salts (typically less than is changed from one content in an organic mod-
100 mmol/L) should be used when organic sol- ifier to another one. The change in the concentra-
vents are to be added to the aqueous buffer. tion of the organic modifier in a period of time is

indicated as gradient slope. For a linear change in When n is larger than 1, the curve of increase
concentration, the gradient slope can be defined “does not hold water,” and when n is between
by the following expression: 0 and 1, the curve of increase “holds water.” In
some chromatographic systems, there are used
ðC2 C1 Þ
D¼ (2.1.2) both types of gradient variation and a “type of
ðt2 t1 Þ curve” can be selected from the software control-
Some HPLC pumping systems allow both a ling the HPLC instrument. As an example, for the
linear change in the gradient and a nonlinear instruments manufactured by Waters (Waters
modification of the concentration (e.g., Corp., Milford, USA), Eq. 2.1.4 is used for curves
Ref. [12]). This change can be achieved using a 2 (n ¼ 8), 3 (n ¼ 5), 4 (n ¼ 3), and 5 (n ¼ 2), and
variation in concentration as a function of time Eq. 2.1.3 for curves 6 (n ¼ 1), 7 (n ¼ 2), 8 (n ¼ 3),
given by the following expression: 9 (n ¼ 5), and 10 (n ¼ 8). These curves are shown
in Fig. 2.1.4 for t1 ¼ 0, C1 ¼ 0, and t2 ¼ 5, C2 ¼ 100.
t t1 n Much less frequently than reciprocating
CðtÞ ¼ C1 þ ðC2 C1 Þ (2.1.3)
t2 t1 pumps, syringe pumps are sometimes used in
where n is larger than 1 for curves of increase HPLC. Only the recent developments in UPLC
that “holds water,” and is between 0 and 1 for made the syringe pumps more attractive.
“does not hold water” type of curve. A different UPLC requires lower flow rates (of the order
type of nonlinear curve can be obtained using a of 0.2e0.5 mL/min) and uses less solvent
variation in concentration with a function given compared to conventional HPLC [13]. In syringe
by the following expression: pumps, a cylinder is loaded with the mobile
phase that is delivered at a specified flow rate
t2 t n by the movement of a piston. The flow from a sy-
CðtÞ ¼ C2 ðC2 C1 Þ (2.1.4)
t2 t1 ringe high pressure pump can be virtually
100
90
1
2
80
3
70 4
5
Composion %
60
6
50
40 7
8
30 9
20
10
10
11
0
0 1 2 3 4 5
Time (min)
FIGURE 2.1.4 Gradient variation curves using both Eqs. 2.1.3 and 2.1.4 used in Waters instruments.

fluctuation free as compared to that from a recip- volumes after the chromatographic column also
rocating pump. The price of a syringe pump can play a role in potential peak broadening.
also be lower. Pumping system for micro- and Another factor contributing to dilution and
nano-HPLC uses specific procedures such as modifications in sample plug shape are the
splitters of an initial higher flow [14]. “void spaces” in fittings that connect the injector
and the chromatographic column. Loss of resolu-
tion by peak broadening due to large void spaces
Tubing and connectors and also due to turbulent flow either along the
After the high pressure pumps, the tubing uti- tubing or in fittings must be avoided. The fittings
lized for connecting different modules of the typically use a nut that connects to a port and a
HPLC has special requirements [15]. These tubes ferule used to secure the end of the tubing in
that have a small internal diameter must be inert the fitting port. Common connector parts, correct
and also must withstand the high pressure fitting, and incorrect fitting of tubing with for-
generated by the pumps. Typical materials for mation of a void volume are shown in Fig. 2.1.5.
the tubing are stainless steel (316 stainless steel)
and PEEK. Stainless steel is inert in most sol-
Injectors and autosamplers
vents, while PEEK may become very stiff after
using solvents such as tetrahydrofuran or dime- The role of the injector is to add in the mobile
thylsulfoxide. Also, stainless steel tubing can be phase a small, precisely measured volume of a
used even at very high pressure, while some re- solution containing the sample. The injection
strictions to pressure are applied to the PEEK must be done reproducibly and accurately.
tubing (as function of internal diameter). How- Reproducibility of injection is of particular
ever, for IC chromatography, PEEK is the mate- importance, and modern injectors typically
rial of choice for tubes and connectors. Tubes of show less than 0.5% RSD% in the injected vol-
several internal diameters (i.d.) are available, ume. The accuracy errors in injection volume
such as 0.12 mm i.d. (0.005 in), 0.17 mm i.d. are important mainly when comparing different
(0.007 in), 0.25 mm i.d. (0.010 in), 0.50 mm i.d instruments since for the same instrument the
(0.020 in), etc. (for both stainless steel tubing use of standards for quantitation may compen-
and PEEK tubing, a color code is available to sate small variations from a nominal volume.
designate the i.d.). The choice of the tubing after However, for a specific method, it is recommen-
the injector starts to play a role in the shape of the ded to keep the same injection volume when
sample plug. Tubing with 0.12 mm i.d. (0.005 in) injecting different samples in order to avoid ac-
is in general recommended to connect the curacy problems. One common type of injector
injector with the chromatographic column. This uses a loop of a precise volume which is filled
tubing has a volume of about 0.13 mL/cm such first with the sample and then connected to the
that a sample of 5 mL will spread over about flow circuit using a switching valve. This system
38 cm, diminishing the effects of sample plug allows only the injection of a fixed volume of
shape modifications. The tubing and the void sample, equal with the loop volume. However,
FIGURE 2.1.5 Tubing connection to a port, correct fitting, and incorrect fitting with a void space.

the volume of sample solution to be injected in sample, and loads it in the injector loop. The
different analyses may need to be varied. For sample solution is subsequently placed in the
example, for a very diluted sample or for detec- mobile phase flow as a solution plug.
tion that is not very sensitive, a larger volume Since in modern HPLC systems the autosam-
of sample may be necessary in order to assure pler capability allows the injection of a number
a good measurement. of samples (e.g., 100, 108, etc.) one after the other,
Samples that generate a high detector signal the samples toward the end of the queue may
need to be injected at lower volumes. Conven- stay in the autosampler for a rather long time.
tional HPLC systems have injectors capable to For improving sample stability in time, it is com-
inject between 1 mL up to 100 mL sample solution mon that HPLC autosamplers also have the
depending on the instrument model, typical vol- capability of cooling. This may also help in
umes being between 1 and 20 mL. Special systems reducing evaporation when the sample solvent
have the capability to inject larger volume (up to is volatile and the septum of the sample vial
1000 mL in special systems) or very low volume has been already punctured.
(e.g., 0.1 mL). Injection of different sample vol- The sample is ideally introduced in the mo-
umes can be achieved using a larger loop that is bile phase flow as a zone (plug) with the concen-
only partially filled with sample (partial loop in- tration of the sample following a perfectly
jection). The loop is typically filled initially with rectangular profile. The force pushing the fluid
the mobile phase, and then the sample is intro- through the tube is given by the cross-section
duced in the loop, occupying only a small portion area of the tube multiplied by the pressure
of its volume. The placing of the loop in the mo- drop Dp, or pr2t Dp (where rt is the tube radius).
bile phase main circuit is then done in a similar In a laminar flow, the layer adjacent to the
manner as for fixed volume loops. The sample wall is held virtually stationary by adhesion,
volume is typically controlled using a computer. and the inner fluid layers slide over the ones
For UPLC, the injection volumes can be between closer to the wall creating a shear force. This
20 and 3000 nL, which is smaller than for force is given by the following expression:
common analytical HPLC, and for semiprepara-
du
tive or preparative HPLC the volumes can be Fh ¼ h A (2.1.5)
much larger. These systems may need specially dr
designed injectors. where h is the liquid viscosity, u is the fluid
Injector systems with automation capability linear velocity, dr is the infinitesimal distance be-
are common (computer controlled autosam- tween the layers, and A is the area of contact be-
plers). In autosamplers, the samples are typically tween the layers of the fluid [16]. The two forces
placed in a tray containing capped vials with (the force pushing the fluid through the tube and
septum (e.g., 2 mL vials) each one containing a the shear force) being equal in a steady flow, at a
solution of the sample. From a large number of distance r from the center of the tube and taking
samples in different vials (or well plate), these A ¼ 2prL (L is the length of contact between
automatic systems have the capability to select layers), the following relation should be
the desired sample vial from the tray, and to satisfied:
repeat the injection at a specified time or upon
du Dp
receiving an electrical signal from the computer. pr2 Dp ¼ 2prLh or du ¼ rdr
For injection, a needle of the injector penetrates dr 2Lh
the vial septum, draws the desired volume of (2.1.6)

For a tube with radius rt, by integration, Eq. connection with the nature of sample solvent in
2.1.6 gives the fluid velocity at any point at dis- Section 7.6.
tance r from the tube center: In autosamplers, since several samples are
injected one after the other, it is possible to see
Dp 2
uðrÞ ¼ rt r2 (2.1.7) carryover effects. Carryover effects represent
4Lh
the contamination of a sample with small
Eq. 2.1.7 shows that due to the friction with amounts of components from the previous sam-
the tubing walls of a viscous fluid, downstream ple that remained in the autosampler after an in-
of injector (in a laminar flow), the shape of the jection. This problem is typically solved using a
sample plug is changing and generates a para- needle wash. Some autoinjectors have the capa-
bolic profile. This process, as well as the diffu- bility of mixing the sample with specific reagents
sion and other convection effects, contributes to from different vials, in case derivatization is
deviations of the chromatographic peak from necessary before the separation and detection
the ideal Gaussian shape, introducing tailing of analytes. Also, cooling and heating capability
and asymmetry (see Section 3.1). is frequently present in modern autoinjectors.
Two important parameters must be selected Usually, the injection operation is unselective.
by the operator regarding injection: (1) the na- However, special on-line sample preparation
ture of the solvent for the sample and (2) injec- methods require more special injection tech-
tion volume. Besides the obvious requirement niques. In case of a multidimensional HPLC,
that the solvent for the sample should dissolve for example, a fraction from eluted sample
it completely, this solvent must also be soluble from a first column can be transferred by an inter-
in the mobile phase. The injection volume is face to become an injected sample to a second col-
selected depending on a number of factors umn. Injection devices normally do not affect
including the type of instrumentation (HPLC, retention parameters unless operation problems
UPLC, detector type, etc.), the sensitivity of the occur (e.g., Refs. [17e19]). Also, in most HPLC
detector, the loading capacity of the column applications, one injection is done for each chro-
(maximum amount of sample that still allows matographic run. However, multiple injections
separation) (see Section 3.4), the effect of sample in a single run (MISER) are possible for specific
solvent on peak shape (see Section 7.6), etc. analyses, such that a larger number of samples
There are problems with both too small volumes can be analyzed within a specific interval of
and with too large volumes of injection. Too time, and with a lower solvent usage [20].
small volumes may lead to problems with injec-
tion reproducibility, sensitivity of the detector,
Column holders
or sample loss in the chromatographic column,
but offer better peak shape sometimes resulting The column in modern HPLC systems is usu-
in better separation. Too large volumes may ally placed in a column holder that may also play
affect the peak shape (broadened, with flat top, the role of a column heater/cooler. Common col-
asymmetrical) that affects separation. A large umn holders have the dimensions capable to
volume of sample does not necessarily mean a accommodate one, two, or even more chromato-
large amount of analyte (e.g., when the sample graphic columns. The column is kept in the col-
is very diluted), but when the large volume is umn holder at a specific temperature that can
also associated with too much analyte, this can be set, usually at a value from 5 C (9 F) to
be associated with an overload of the column 65 C (149 F), different instrument vendors offer-
(problems with the separation) and of the detec- ing column holders capable of achieving a spe-
tor (leading to a nonlinear response). Further dis- cific temperature range. For some separations,
cussion on injection volume will be made in the column may be kept at ambient temperature,

while in some other studies the column temper- (microfluidic chips) are also available as con-
ature is an important parameter to consider. tainers for the stationary phase. Based on the in-
Most column holders are also equipped with a ternal diameter of the analytical column, they
leak detector. Most modern HPLC instruments are sometimes classified in the literature as
utilize thermoelectric Peltier technology in order (1) standard (3.0e4.6 mm i.d.), (2) minibore
to heat or cool the separation column. Peltier ef- (2.0e3.0 mm i.d.), (3) microbore (0.5e2.0 mm
fect is a temperature difference created by i.d.), (4) capillary (0.2e0.5 mm i.d.), and (5)
applying a voltage between two electrodes con- nanoscale (0.05e0.2 mm i.d.). Larger columns
nected to a sample of semiconductor material. are used for semipreparative and preparative
For HPLC working at elevated temperature purposes. Capillary columns represent a special
(indicated as high temperature HPLC), the mobile type as they are made from a silica capillary
phase needs to be preheated before entering the that contains the stationary phase. Capillary
column in order to achieve maximum efficiency. columns on a chip were also experimentally
For such special technique, the column heater made. The empty volume of the column can be
temperature can be maintained at a specified tem- easily calculated as the volume of a cylinder
perature up to 200 C [21] (see also special HPLC V ¼ (p/4)d2L, and for analytical columns, V
setups). Others custom-made column holders are ranges between 0.02 and 20 mL.
available for specific applications [22]. The nature of the stationary phase is selected
based on the type of chromatography that is uti-
Chromatographic columns lized for the separation (normal phase, reversed
phase, ion exchange, size exclusion, etc.). A large
The chromatographic column is designed for assortment of types of stationary phases (column
performing the separation in HPLC. Its role packings) is available. The stationary phase usu-
and properties are related to various subjects dis- ally consists of small solid particles with special
cussed in this book, and in the present section is properties. Besides small particles, porous poly-
given only a simplified overview of the subject meric materials, and also monolithic materials
(see Chapter 8). The column typically consists can be used as stationary phase. When the sta-
of a tube made from metal (stainless steel) or tionary phase is made from small particles, the
plastic (e.g., polyetheretherketone, PEEK) that particles should have specific physical and
is filled with a stationary phase. At the two chemical characteristics to serve as a stationary
ends inside the column are special frits that phase. The surface area of particles is one of
keep the stationary phase from moving, and the most important physical characteristics, be-
outside are fittings that allow the connection ing directly related to the retention in the column
with high pressure tubing. The physical dimen- of the compounds to be separated. The effect on
sions of common analytical chromatographic separation of the particle size is also important.
columns vary, and values for length (internal) Particle size influences in particular the eddy
L can be between 30 and 250 mm (common diffusion, which appears when local small
length 50, 100, 150, 250 mm), and internal diam- streams of liquid follow different channels in
eters d can be between 1 and 10 mm (common di- the column. The effect is common within porous
ameters 2.1, 3.0, or 4.6 mm). Other dimensions particles. This generates a broadening of the
are possible, particularly when the column is chromatographic bands (discussed in Section
designed for special tasks. The newer columns 3.1), which is not a desired feature. When the sta-
have the tendency of being shorter and nar- tionary phase is made from small porous parti-
rower, as the solid particles that form the station- cles, these particles are frequently obtained
ary phase are made smaller. Special cartridges from an inert substrate material (such as silica)

that is covered with the active phase. The parti- (3) surface reactivity, (4) density and distribution
cles can be of three main types: porous, superfi- of surface reactive centers, etc. The subject of the
cially porous (core-shell), and pellicular. Porous nature of stationary phases used in various ana-
particles are still the most common type of sta- lyses is fully discussed further in this book (see
tionary phase used in HPLC. They are made Chapter 8). In some systems, more than one
from particles usually of 1.7e5 mm diameter chromatographic column is necessary for
with a specific porous structure (e.g., of silica) achieving the desired separation. In size exclu-
and with the surface of this structure covered sion chromatography, for example, two to four
with an active constituent. This constituent can columns may be connected in series. In other
be physically or chemically bonded on the solid types of separation, the use of more than one col-
inert support, the bonded phases being the umn is less common. The nature of the columns
most common type. The column dimension used in series may be the same or may be
and the diameter of the particles used as station- different. More than one column is also used in
ary phase determine if the separation is consid- multidimensional HPLC, where a portion of
ered HPLC or UPLC and the adequate the initial separation is further submitted for a
instrumentation is selected. The use of UPLC is second separation in a different column (usually
necessary for columns with smaller diameter selected as “orthogonal”).
d and with smaller particles with diameter dp of Some chromatographic columns require a spe-
1.7 or 1.8 mm. Since reverse-phase (RP-HPLC) is cific temperature for performing a good separa-
the most utilized technique, the largest variety tion, and for this purpose special column
of columns is of RP type. These columns have a holders are used. Common column holders
hydrophobic active phase, for example, with have the capability to control the column temper-
octadecyl groups (C18) or with octyl groups ature in a range from about 10 C below ambient
(C8) bonded on silica. For other types of chroma- to 60e70 C. Higher temperatures can be achieved
tography, the stationary phase may be made in with special ovens used in high temperature
various forms. For example, ion exchange HPLC. In addition to heating the column, the col-
HPLC can use particles from a substrate inert umn holders typically are able to heat the solvent
material that are covered with the active phase entering the column, since peak shape distortions
but also various types of ion exchange poly- may be noticed when the column and the
mers. Size exclusion chromatography typically entering solvent have different temperatures.
uses perfusion particles made from silica or In order to protect the stationary phase from
special types of polymers. These particles the analytical HPLC column, it is common to
contain very large pores (400e800 nm) con- use small pore frits (e.g., with 0.45 mm pores) as
nected with a network of smaller pores well as guard columns (cartridges) in the path
(30e100 nm). The structural rigidity of these of the mobile phase before the column. The frits
particles is not as good as that of common are usually made from stainless steel or titanium
porous particles made from silica, and restric- and filter the mobile phase. When the mobile
tions to the maximum pressure that can be phase is acidic, the metallic surface of the frit
used with the columns made with these parti- can adsorb acidic solutes which influence their
cles are typically indicated by manufacturer. recovery or lead to unexpected peak tailing.
At higher pressures than recommended, the The covering of metal frit surface with hybrid
stationary phase may “collapse” and the col- organic/inorganic materials mitigates analyte-
umn can be damaged. to-metal interactions and improves the recovery
The chemical properties of the particles form- of acidic analytes [23]. For column protection,
ing the stationary phase include (1) chemical na- more important than frits are the guard columns.
ture of the active surface, (2) chemical stability, The guard (cartridge) columns are selected to

match the stationary phase of the analytical col- detector (electric) output. Other disturbances in
umn (same active material), but their length is the detector signal may include long-term noise
much shorter (e.g., a few mm), and in some cases and drift. The long-term noise appears as a fluc-
their stationary phase has larger particle size. In tuation of the signal with a wider frequency. The
the analyzed samples, there are sometimes com- drift is a continuous variation (increase or
ponents that are very strongly retained by a spe- decrease) of the signal for a period of time com-
cific stationary phase. These components do not parable with the length of the chromatogram.
elute and tend to accumulate at the head of the col- Drift may be caused not by the detector itself,
umn, deteriorating its performance in time. The but by changes, for example, in the composition
use of a guard column allows selective retention of the mobile phase during gradient elution.
of these components without affecting the effi- The qualities of the detectors should include
ciency of the column, retention time, backpressure, the following: (1) capability to provide quantita-
or the level of analytes. Guard columns are tive or both qualitative and quantitative informa-
changed from time to time, while the analytical tion, (2) good selectivity, (3) good sensitivity, (4)
columns have longer service life (see Section 8.3). reproducible response, (5) large dynamic range,
(6) linearity in a wide range of concentrations of
General comments regarding detectors sample, (7) low baseline noise and no baseline
drift, (8) capability to make detection in a small
In HPLC, the mobile phase and the eluted ana- volume of sample, (9) capability to not contribute
lytes pass through a detector capable of perform- to peak broadening, (10) stability to changes in
ing measurements. These are based on the fact flow and environmental parameters, and (11)
that the molecules of the sample have physico- high frequency of data collection.
chemical properties different from that of the Some detectors have the capability to generate
mobile phase. The measured properties are deter- only quantitative information, while others offer
mined by the nature of the compound to be both qualitative and quantitative information,
analyzed and that of the mobile phase. The choice such as the MS detectors. Although the response
of a specific property for detection depends on fac- of the detectors used in HPLC is typically not
tors such as the extent of difference between that of diagnostic for the chemical nature of the analyte,
the analytes and of the mobile phase, sensitivity depending on the analyte and sample character-
of the detector to the specific property, availability istics, the identification of compounds is possible
of the detector, etc. The selection of a specific detec- using liquid chromatographyemass spectrom-
tor is also correlated to the separation conditions etry (LC-MS), liquid chromatographyemass
used for the analysis. Some detectors have specific spectrometry/mass spectrometry (LC-MS/MS),
requirements for the nature of mobile phase, selec- and in some cases even other detectors.
tion of isocratic or gradient separation, selection of The selectivity of the detector is related to the
a specific temperature for the column and mobile response to only a specific type of analyte. How-
phase, etc. [24]. ever, some detectors are designed to respond to
The detector response (output) is typically all analytes (such as the RI detector, or ELSD)
dependent on the instantaneous concentration and are indicated as universal detectors. When
(or amount) of the detected species, and for this the HPLC separation is good, the universal de-
reason the quantitation is based on the area of tectors are very useful since they account for all
the chromatographic peak (see Section 3.4). The the components. Selective detectors respond to
output appears as an electrical signal recorded only a class of compounds or they can be set to
in the form of a chromatogram. In addition to detect only specific compounds. The detector
the signal, all detectors are affected by the selectivity adds one more capability for detecting
“noise,” which is the random oscillation of the separately certain analytes, and this can be a

significant advantage when the HPLC separa- The dynamic range of a detector is related to
tion is not possible (or is incomplete). the range where the detector has a response
Detector sensitivity is a very important factor dependent on the analyte amount or concentra-
in HPLC analysis. This sensitivity depends on tion. The detector may not have a linear response
several factors such as analyte properties, sam- for the entire dynamic range, but even in the
ple matrix, mobile phase properties and also on range where the response is not linear, a positive
detector settings (e.g., electronic amplification dependence should exist between the signal
of the signal), and detector manufacturer. For versus the amount of analyte. The dynamic
these reasons, in analytical methods using range of a detector can be independent of the na-
HPLC, parameters such as limit of detection ture of the analyte (for universal detectors), but
(LOD) and limit of quantitation (LOQ) are re- also can be highly dependent on the analyte na-
ported (see Section 3.4). They characterize glob- ture. The dynamic ranges of several types of de-
ally the sensitivity of the method and consider tectors are given in Table 2.1.1.
a number of factors, including the detector sensi- Detector linearity indicates the range in which
tivity. The range of sensitivities of various detec- the dependence of the detector signal depends lin-
tors are indicated in Table 2.1.1. early on the amount or of concentration of the an-
Depending on the injected volume of the sam- alyte. Similar to the detector dynamic range, the
ple, the concentration of the analyte may vary in linear range of a detector can be either indepen-
the injected solution. A typical injection volume dent of the nature of the analyte or highly depen-
of 10 mL would require 100 times higher concen- dent on this nature. The typical linear ranges for
trations per mL compared to the values indi- various detector types are given in Table 2.1.1.
cated in Table 2.1.1. The low baseline noise and no baseline drift
Reproducibility of the response to the same are properties typically related to the quality of
injected sample is an important characteristic of the electronic parts of detector, or the quality of
any detector. Reproducibility of a detector is other detector components (e.g., life time of the
mainly related to the quality of detector con- UV lamp, dirty flow cell) or due to the air bub-
struction. Since HPLC is mainly used for quanti- bles trapped inside the flow cell.
tative analysis, the reproducibility of the detector The capability to make detection in a small
response is a very important parameter. volume of sample is usually related to the
TABLE 2.1.1 Sensitivity (in amount) for various types of HPLC detectors and their typical dynamic and linear ranges.
Minimum mass Minimum mass Dynamic range of Linear range

Type of detector injected (typical) injected (extreme) concentrations concentrations
UV-Vis spectrometry 0.1e1 ng 0.01 ng 10þ5 10þ4

Fluorescence spectrometry 1e10 pg 10 fg 10þ4 10þ3
Refractive index 100 nge1 mg 10 ng 10þ4 10þ3
Electrochemical amperometric 0.1e1 ng 100 fg 10þ4 10þ3
Electrochemical conductometric 0.5e1 ng 0.5 ng 10þ4 10þ3

Mass spectrometry 1 pge1 ng 10 fg 10þ4 10þ3
Evaporative light scattering 5 mg 0.5 mg 10þ3 10þ2
FT-IR spectrometry 1 mg 0.5 mg 10þ3 10þ3

volume of a flow-through cell or other parts in Most modern detectors used in HPLC do not
the instrument construction. For UV-Vis detec- generate a continuous signal based on measured
tors, for example, instrument manufacturers physico-chemical property. The signal generated
may offer cells of different dimensions, with by the detector is typically obtained at short time
longer path of the beam of light in the flow- intervals, and a measurement frequency (some-
through cell for achieving higher sensitivity of times indicated as sampling frequency) is charac-
the instrument, but with slightly larger cell vol- teristic for each detector. It is possible in some
ume, or cells with shorter path for the light instruments that such frequency can be set by
beam, but also with smaller volumes. Microcells the operator, but in other detectors this fre-
are also available for certain instruments that are quency is fixed. It is important for the measuring
designated for micro-HPLC systems. Special frequency of the signal to be high enough for
cells are also necessary when using capillary obtaining an accurate representation of chro-
HPLC columns and low flow rates. matographic peak. A sampling frequency of
The capability to not contribute to peak about five points per second could be sufficient
broadening is also related the HPLC construc- for peaks with 4e5 s width (or larger). However,
tion. The absence of dead volumes, small flow- for narrow peaks, the sampling frequency must
through cells, laminar flow within the detector, be higher, such as 20 or 50 Hz. The measurement
and adjustment of the total volume of mobile frequency must be high in particular for narrow
phase in the detector in accordance with the chromatographic peaks. The curve representing
range of injection volume for the sample are the peak shape is generated by connecting each
some of the steps toward achieving little or no measurement point, and sparse points do not ac-
contribution to peak broadening [25]. count properly for this shape and introduce er-
The stability of the detector response to rors, in particular regarding peak areas.
changes in flow of mobile phase may be an It is common in modern HPLC systems that
intrinsic characteristic. For some detectors such they have more than one detector available. For
as UV-Vis, the response is more stable to changes example, UV-Vis and fluorescence detectors are
in flow rate since this detector responds to frequently coupled in series, although not neces-
instantaneous analyte concentration. For other sarily used simultaneously. Since the flow
detectors, the stability cannot be achieved through a detector may pose some backpressure,
without a constant flow since they are respon- when using detectors coupled in series, it should
sive to the amount of analyte reaching the detec- be verified that the flow-cell of the detector up-
tor and not to the instantaneous analyte stream can handle the backpressure generated
concentration. For example, for MS detection, by the second detector. For the detector down-
the response is dependent on the amount of an- stream, it should be verified that undesirable
alyte in the source and therefore is dependent peak broadening does not occur because of the
on the flow rate. In addition, the dependence of up-stream detector. The coupling of detectors
response on flow rate in MS is not linear, and in parallel is also possible, but care must be taken
higher flows beyond an optimal point may be to assure that appropriate flow goes through all
in some cases detrimental to sensitivity. Other the detectors. Since different detectors may pose
detection techniques, such as RI are also flow different backpressures to the flow, the risk ex-
dependent. In RI, the dependence is related to ists that most (or even all) of the flow goes to a
baseline stability. The stability of a detector to single detector. A short discussion about the
changes in flow must be known before modi- main detection techniques in HPLC is further
fying the flow rate. presented.

UV-Vis spectrometric detectors defined by the logarithm in base 10 of the inverse

of transmittance as follows:
This type of detectors are basically in-line UV-

Vis spectrometers equipped with a flow through 1 I0
cell. They can be classified as fixed wavelength, Al ¼ log ¼ log (2.1.9)
T II
variable wavelength, and photodiode array
UV-Vis detectors. In these instruments, a beam Absorbance is related to the molar concentra-
of monochromatic light (more correctly a beam tion CX (also noted interchangeably as [X]) of the
of light with a narrow wavelength range) is absorbing species X by LamberteBeer law:
sent through the eluent flowing through a cell
Al ¼ εl ½XL (2.1.10)
with two quartz windows or lenses of small vol-
ume (e.g., 1 mL for a microcell, 5 mL for a semi- where εl is the molar absorption (absorbance) co-
micro, and 14 mL for a standard cell, with path efficient at the specific wavelength l and L is the
length of 5e10 mm depending on the cell, path length of the light through the sample. For
some cells having a conical shape). The typical quantitation purposes, the absorbance is
source of light in a UV-Vis detector is a low- commonly used, because it is proportional with
pressure arc discharge deuterium lamp with the concentration. The quantitation can be done
light energy in the range 190e600 nm. Some in- using calibration curves or standard addition
struments may have an additional tungsten fila- method [27]. The absorbance of the liquid eluting
ment lamp for extending the visible range. The from the separation system is typically measured
monochromator consisting in a movable diffrac- at specific (small) time intervals (not continu-
tion grating (or prism) for light dispersion that ously), generating points that form the chro-
can be rotated allows the selection of light of a matogram. The peaks in the chromatogram
set-up wavelength through an exit slit. The indicate an increase in the absorbance Al when
monochromatic beam is split (using a beam an absorbing species elutes from the chromato-
splitter), one part going through the cell with graphic column. For variable wavelength detec-
the sample and to the detector, and the other to tors, the wavelength can be selected (by
a reference detector. The baseline is obtained rotating the grating) and is usually set where a
from the reading for the eluate when no solute strong absorption of light by the analyte occurs
is emerging from the column [26]. (possibly at the maximum). This type of detector
Two related quantities, transmittance T and is one of the most commonly used in HPLC.
absorbance A, are measurable for the light pass- Older detectors were made to measure the absor-
ing through the solution. Transmittance is bance at a fixed wavelength such as 254 nm. This
defined as follows: wavelength corresponds to the maximum emis-
sion (253.7 nm) of a mercury lamp that has
T ¼ II =I0 (2.1.8)
been used as a UV source in simpler spectropho-
In Eq. 2.1.8, Io is the intensity of the radiant en- tometers. The area under the chromatographic
ergy incoming to the sample and I1 is the inten- peak of the analyte being proportional with the
sity of the emerging light (T can also be analyte amount injected into the column is
expressed as percent). As expected, T is a func- used for quantitation with the help of a (linear)
tion of the wavelength l (or of frequency calibration curve or a response factor between
n ¼ clight/l, where clight is the speed of light) of peak area and the analyte concentration. The
the radiation that is absorbed. Absorbance Al is peak height can also be used for quantitation

assuming equal peak widths for all concentra- such as collisions with other molecules. In this
tions. Some UV-Vis detectors have the capability case, the electron may go to another excited elec-
to capture the absorbance for a whole spectrum tronic state with lower energy and then, emitting
range (UV-Vis diode array detectors or DAD). a photon, reaches the ground state. It is also
If the entire UV-Vis spectrum is measured in possible that no intermediate electronic state is
different points across a chromatographic peak, present, but the molecule acquires a lower vibra-
this can be used for the evaluation of peak purity tional energetic level and jumps to the ground
and also can be a guide for qualitative identifica- state by emitting a photon of lower energy
tion of the analyte, although UV-Vis spectra are than the absorbed one. In both these cases, the
very seldom sufficient for compound identifica- fluorescence radiation has a lower frequency
tion. The UV region of spectrum starts at about than the excitation radiation.
190 nm, and modern UV-Vis instruments have Fluorescence by emission of radiation at
a working range between 190 and 600 nm. How- higher frequency than the absorbed one is also
ever, the common range of practical utility in UV possible (anti-Stokes radiation), but it is uncom-
spectrophotometric measurements starts at mon. The average lifetime of an excited state of a
about 205 nm or higher. At lower values than molecule M* undisturbed by collisions is about
this wavelength, a strong light absorption usu- 108 s, and fluorescence can take place within
ally takes place because the solvents used as this length of time. For some special compounds,
mobile phase start absorbing. The wavelength the molecules can remain for a longer time in a
cut-off of various solvents can be found in tables metastable excited state. In this case fluorescence
(e.g., Ref. [28]). Depending on the nature of the can be observed long after the initial radiation is
analyzed material, the detection limit of the interrupted. This type of fluorescence is
UV-Vis detection in HPLC can be 0.1e1.0 ng, commonly called phosphorescence. Fluores-
with a linear range of five orders of magnitude. cence is less frequently observed than expected
With an appropriate solvent that does not absorb because it is very common that only nonradia-
in the range of UV-Vis measurement, the use of tive loss of energy takes place. The theory of
elution gradient can be applied for separation. fluorescence emission shows that the intensity
of fluorescence Fint at the emission wavelength
Fluorescence and chemiluminescence l2 can be expressed as a function of the intensity
detectors I0 of the excitation radiant energy with wave-
length l1 incoming into the sample, by the
Fluorescence (FL) is the process of emission of
following expression:
light by a molecule after absorbing an initial ra-
diation (excitation light). A molecule M goes
from a lower energetic state (commonly ground Fint;l2 ¼ I0;l1 1 exp εl2 ½XL F (2.1.11)
state) to an excited state M* by absorbing energy. In Eq. 2.1.11, F is the (quantum) fluorescence
The emission process may take place by the yield of the process, the other parameters being
molecule bouncing back to the initial state the same as defined for UV-Vis. For low concen-
without the change in the wavelength of the trations, 1exp (εl2 [X] L) z εl2 [X] L, and the
absorbed light. In this case, the process is diffi- intensity of fluorescence Fint,l2 is related to the
cult to use for analytical applications. However, concentration [X] by the approximation relation:
it is possible that part of the energy of the excited
molecule M* is lost by nonradiative processes Fint;l2 ¼ I0;l1 εl2 ½XLF (2.1.12)

In reality, only a part of emitted fluorescence interfering molecules, quenching produced by

is measured in the analytical instrument oxygen dissolved with the solvent, etc. Because
and the intensity of this measured fluorescence the intensity of fluorescence increases linearly
F0 int,l2 is given by the following expression: with the intensity of the initial radiation, laser-
induced fluorescence (LIF) detection is a success-
F0 int;l2 ¼ a I0;l1 ½X (2.1.13)
ful technique applied in HPLC. For HPLC, lasers
where a is a constant coefficient that incorporates are a convenient excitation source because they
all the constants including the losses due to par- have intense light focused into a small volume,
tial fluorescence measurement. Measurement of they are highly monochromatic, and the associ-
fluorescence intensity (usually at the maximum ated Raman light has a well-defined wavelength
of the emission band) is the base of quantitation that can be avoided with the monochromator
of the fluorescent species. In practice, the fluores- used for observing fluorescence. However, LIF
cence intensity is measured using sensitive light is still affected by background interference
detectors (FLD) that generate an electrical signal commonly arising from the Raman effect in the
of intensity depending on a calibration constant blank (molecular scattering) or from low level
for the instrument. The output is given in lumi- of solid impurities in the solvent producing Ray-
nescence (or light) units (LU) that are arbitrary leigh light scattering. The use of fluorescence
units proportional with the fluorescence intensity, detection (FLD) in HPLC is common. Detectors
but specific for the measuring instrument [29]. with constant excitation wavelength and vari-
Similar to UV-Vis detection in HPLC, the fluo- able absorption or with variable wavelength
rescence is measured in a flow-through cell that excitation and absorption are commercially
is connected to the flow of the eluent incoming available. Depending on the nature of the
from the chromatographic column. Modern fluo- analyzed material, the detection limit using
rescence detectors may have the capability of FLD can be as sensitive as 102e103 ng/mL,
recording a tridimensional emission spectrum with a linear range of four orders of magnitude.
of the analyte by stopping the mobile phase When appropriately selected, the use of elution
flow at a chosen retention time (selected for the gradient can be applied for separation without
analyte) and performing a scan of the entire interfering with the fluorescence. Different fac-
UV range used for excitation and for the entire tors related to the mobile phase influence fluo-
emission band. In this way, the tridimensional rescence such as pH, solvent nature,
fluorescence spectrum is displayed as a depen- temperature (as much as 2%), presence of impu-
dence of fluorescence intensity on emission rities, as well as the flow rate.
wavelength and excitation wavelength. It is Chemiluminescence (CL), another technique
common in modern fluorescence HPLC detec- used for HPLC detection, is the emission of light
tors that the excitation beam is generated by a as a result of a chemical reaction. Certain com-
high power lamp that flashes at a specific num- pounds achieve excited energy states in specific
ber of times per second (e.g., 296 times in an Agi- chemical reactions and emit light following a
lent 1200 Ser. detector), such that the signal is transition to ground state. The wavelength of
modulated in time. Also, the systems typically the light emitted by a molecule in chemilumines-
have a reference detector that measures the cence is the same as in its fluorescence, the en-
excitation light and corrects the flash lamp ergy levels of the molecules being the same.
fluctuations. The detection in fluorescence The difference comes from a different excitation
methods encounters several difficulties process. If the energy of the chemical reaction
because of nonlinearity of fluorescence due to is lower than required for attaining the excited
self-absorption effects, difficulty in discrimi- state, the chemiluminescence does not occur.
nating between overlapping broad spectra of Also, the deactivation of the excited molecule

by nonradiative processes such as collisions with location of the beam on the (photoelectric) detec-
other molecules takes place for chemilumines- tor is made to reduce the detector output propor-
cence similarly to fluorescence. The chemilumi- tional to the deviation of the beam from the
nescence intensity follows the same law as reference position. This output is electronically
fluorescence with the difference that quantum modified to provide a signal proportional to the
yield F from fluorescence must be replaced concentration of the solute in the sample cell.
with a different quantum yield FCL, which is The refractive index depends on the wave-
defined as the proportion of analyte molecules length of the incident beam, and the most accu-
that emit a photon during chemiluminescence. rate RI measurements are done with
The FCL increases with the efficiency of the monochromatic light (usually 589 nm, the so-
chemical reaction producing the excitation dium D line). With optical corrections, white
(such as the oxidation process). Higher energies light can still be used for the measurements.
required by molecules to achieve the excited RID is a universal detector that can be utilized
state diminish FCL. In analytical uses of chemilu- without the need for chromophore groups, fluo-
minescence, one more factor that must be taken rescence bearing groups, or other specific prop-
into account is the time frame of the light emis- erties in the molecule of the analyte. In many
sion. Certain chemiluminescent systems, cases, the sensitivity of RI detection is, however,
although with very good FCL, may emit the light not as good as that of other types of detection.
for a period of 40e50 min. Much shorter times Also, it is not possible to use elution with
can be achieved using a catalyst. Because no exci- gradient for the mobile phase, since this is asso-
tation light is needed in chemiluminescence, the ciated with large variations in the refractive in-
interfering light from Raman effect or light scat- dex of the mobile phase. Refractive index is
tering by trace particles is nonexistent. In addi- sensitive to temperature changes, and a constant
tion, the development of detectors virtually temperature must be maintained during mea-
able to detect single photons makes the tech- surements. The response of the detector is given
nique highly sensitive. Concentrations as low in arbitrary RI units, depending on the detector
as a few hundred amol/mL of material were settings, but proportional with the concentration
detected using chemiluminescence for certain of the analyte.
analytes [30]. However, the luminescent mole-
cules are not very common and usually they
are obtained by postcolumn derivatization with
Electrochemical detectors
proper reagents [31]. Various types of electrochemical analytical
techniques can be adapted for HPLC detection.
Among these are amperometric, coulometric,
Refractive index detectors potentiometric, and conductometric techniques.
Refractive index detection (RI or RID) is The techniques more commonly applied in
another common technique in HPLC. Due to the HPLC are the conductometric, amperometric,
modification of the refractive index of a solution and to a lesser extent coulometric procedures
as a function of the concentration of the solute, [32e34]. These techniques can have very high
RI can be used for the quantitation of a variety sensitivity, and the price of the detectors is rela-
of analytes. This detector is based on the devia- tively low. In ion chromatography, conducto-
tion of the direction of a light beam when passing metric measurements are the most common. In
under an angle from one medium to a medium amperometric techniques, the current intensity
with a different refractive index. This deviation is measured in an electrochemical cell when a
depends on the difference in the refractive index specific potential E is applied between two elec-
between the two media. The change in the trodes. Usually, only the reaction at one

electrode is of interest, and a cell can be not indicating the charge nþ) to generate the
composed of a working electrode coupled with compound Red is written as follows:
a nonpolarizable electrode (one that does not
Ox þ n e %Red (2.1.14)
modify its potential upon passing of a current).
0
This is known as the reference electrode. More The electrode potential E for this half-
frequently, a three-electrode cell arrangement is reaction is reported to the potential of a reference
used. In this arrangement, the current is passed standard hydrogen electrode (NHE), which is
between a working electrode (made, for taken as zero. Experimental measurements are
example, from glassy carbon) and an auxiliary commonly done with a saturated calomel elec-
electrode, while the potential of the working trode (Hg/Hg2Cl2 or SCE) or with an Ag/
electrode is measured relative to a separate refer- AgCl reference electrode. The potential of an
ence electrode. The two types of cells are shown SCE electrode versus NHE is þ0.242 V, and the
in Figs. 2.1.6A and 2.1.6B. potential of an Ag/AgCl electrode is þ0.197 V
Any overall cell reaction comprises two inde- versus NHE. If a compound accepts electrons
pendent half-reactions, and the cell potential can from a standard hydrogen electrode, it is defined
be broken into two individual half-cell poten- as having a positive redox potential, and a com-
tials. The half-reaction of interest that takes place pound donating electrons to the hydrogen elec-
at the working electrode surface can be either an trode is defined as having a negative redox
oxidation or a reduction. A simple reduction re- potential. A high positive E0 indicates an oxidant
action for an oxidized compound Ox (notation and a low negative E0 indicates a reducing com-
pound. The reaction takes place spontaneously
in a cell with a positive resulting potential.
Considering a reversible reduction that has a
very rapid electron transfer, and assuming that
both Ox and Red are soluble species, the molar
concentrations COx and CRed at the electrode sur-
face (x ¼ 0) are governed by Nernst equation:
RT COx ðx ¼ 0Þ
E ¼ E0 þ ln (2.1.15)
nF CRed ðx ¼ 0Þ
where E0 is the standard electrochemical poten-
tial of the half-cell, R is the gas constant (in SI
FIGURE 2.1.6A Schematics of a two-electrode flow-cell. units R ¼ 8.31451 J deg1 mol1), T is the tem-
perature (in K deg.), n is the number of electrons
involved in the electrochemical reaction, and F is
Faraday constant (F ¼ 96485.332123 C mol1).
Tables of standard electrochemical potentials in
specific media are available in the literature
[35] and the half-reactions are expressed as re-
ductions. (Potentiometric measurements based
on Eq. 2.1.15 are also used for the pH measure-
ments where the ratio CRed/COx ¼ [Hþ]).
In amperometric measurements, the current in-
tensity is measured in an electrochemical cell
FIGURE 2.1.6B Schematics of a three-electrode flow-cell. when a specific potential is applied between two

electrodes. For the very rapid electron transfer at Eq. 2.1.21 represents the relation between the
the electrode surface, the rate v of the electrochem- potential E and the current intensity I for a
ical reaction (expressed in mol1s1cm2) is pro- reversible redox reaction with very rapid elec-
portional with the current intensity I and tron transfer at the electrode surface.
inversely proportional with the electrode area Ael: For the current intensity equal to half of the
limiting current, I ¼ 1/2 Ilim, the last term in
I
v¼ (2.1.16) Eq. 2.1.21 is null, and the corresponding potential
nFAel indicated as E1/2 is independent of the concentra-
This rate is determined by the mass transfer in tions of the oxidant or reduced species and it is
solution and is given by the rate at which the elec- specific for a Red-Ox system. This E1/2 potential
troactive species are brought to the surface of the is known as “half wave potential” and for a diffu-
electrode. In the absence of convection (for bulk so- sion controlled process (static), a reversible reduc-
lutions) and of migration under the influence of tion reaction with both Ox and Red species
the electric field, diffusion is the only mechanism soluble and only with the oxidant initially
for mass transfer, and the rate vmass transfer (flux) present in the solution, the variation of the current
can be approximated by the following expression intensity I as a function of the working electrode
(equivalent with Fick’s first law): potential E follows an equation of the form:

vmass transfer ¼ mOx COx COx ðx ¼ 0Þ (2.1.17) RT Ilim I
E ¼ E1=2 þ ln (2.1.22)
nF I
where mOx is a proportionality coefficient called
mass transfer coefficient, and COx is the concen- The graph of Eq. 2.1.22 for a hypothetical
tration of Ox in the bulk solution. The largest reduction with E1/2 ¼ 0.5 V is shown in
rate of mass transfer for Ox occurs when Fig. 2.1.7.
COx(x ¼ 0) ¼ 0. The value of the current in these For the case of the electrochemically active spe-
conditions is called the limiting current Ilim, and cies flowing over the surface of an electrode,
its value is given by the following expression: which is the case of electrochemical detection in
HPLC, the current potential dependence is deter-
Ilim ¼ nFAel mOx COx (2.1.18) mined by a convective diffusion process (not only
The expression for COx(x ¼ 0) can be written by diffusion). This makes the limiting current
now using Eqs. 2.1.16e2.1.18 as follows:
Ilim I
COx ðx ¼ 0Þ ¼ (2.1.19)
nmOx FAel
When the reducing species Red is absent in the
Current I(A)
bulk solution, CRed* ¼ 0, and using a relation E1/2

Ilim
similar to Eq. 2.1.19 for the reduced species, the
expression for CRed can be written as follows:
I
CRed ðx ¼ 0Þ ¼ (2.1.20)
nmRed FAel
With Eqs. 2.1.19 and 2.1.20, Nernst formula 0 -0.25 -0.5 -0.75 -1
2.1.15 can be written as follows: Potential E(V)
RT mOx RT Ilim I FIGURE 2.1.7 The currentepotential curve of a Nerns-

E ¼ E0 þ ln þ ln (2.1.21) tian reaction involving two soluble species and only with
nF mRed nF I
the oxidant present initially. In this example, E1/2 ¼ 0.5 V.

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habían de estar ciegos los que
juegan con ellos. Y todo es
sufridero para con otras
tacañerías que se usan, y la
mayor de todas es cuando meten
dados cargados, que llaman
brochas, los cuales hacen de esta
manera: que á los que llaman de
mayor, por la parte del as hacen
un agujero hueco y allí meten un
poco de azogue, que es muy
pesado, y á los de menor donde
están los seis puntos; y después
tapan el agujero, que es muy sutil,
y encima pintan uno ó dos puntos
para que no se vean, y estos
dados llevan los chocarreros
escondidos, y cuando tienen una
suerte de doce ó trece ó catorce
puntos, echan los dados de
manera que se les caya alguno
en el suelo, y haciendo que se
baxan por él, sacan otro de los de
mayor, que meten en su lugar, y
como está cargado en el as, cae
siempre para abaxo y el seis para
arriba; y de la mesma manera
hacen cuando tienen por suerte
siete ú ocho puntos, que meten
un dado cargado en el seis
porque vaya el as para arriba,
yendo el seis para abaxo, y si es
menester meten dos dados de
esta suerte cargados de mayor, y
cuando tienen suerte de doce ó
de trece, alárganse en el parar y
en el decir, de arte que, no siendo
entendidos, todo el dinero es
suyo. Otros dados hay que llaman
falsos, que son mal pintados
porque tienen dos ases y fáltales
el seis, ó tienen dos seises,
faltándoles el as, y conforme á la
suerte que echan y á la necesidad
que tienen, se aprovechan dellos
metiéndolos en el juego tan bien
como las brochas. Y cuando
juegan á las tablas no penséis
que se descuidan los hombres
desta professión, que lo mesmo
hacen con los dados, y
verdaderamente yo tengo por
malo y dañoso también este
juego, assí por jugarse con
dados, como por ser trabajoso y
mohino. Á todos los otros juegos
podéis levantaros y os toman en
una petrera; habéis de esperar á
que se acabe el juego, perdiendo
á cada mano y cada vez que
echáis los dados sabiendo que se
echa para perder y no para ganar,
y assí es el juego más aparejado
de todos para perder la paciencia,
porque es menester esperar á
que el juego ó el dinero se
acaben. Y aunque yo no os he
dicho de diez partes la una de los
males y trabajos y fatigas y
persecuciones y desasosiegos y
afrentas, menguas y deshonras y
infamia que se siguen del juego,
de lo dicho podréis collegir cuán
perjudicial es, assí para la salud
como para la hacienda y la honra
de las gentes que lo siguen;
porque pocos hay que jueguen,
por ricos y caballeros y grandes
señores que sean, que no les
pese de perder, y muchos destos
se acodician á jugar mal por
ganar, y assí veréis muchas
personas de muy gran autoridad,
y de quien apenas se podría
creer, que hacen malos juegos,
por la buena estima y reputación
en que están tenidos que,
apremiados de la conciencia,
restituyen dineros mal ganados,
de los cuales yo conozco algunos
que lo han hecho.
Bernardo.—¿De manera que
queréis condenar á todos los
juegos del mundo y no dejar
ninguno para recreación de la
vida y para poder pasar la
ociosidad del tiempo?
Antonio.—No digo yo tal cosa,
que otros juegos hay lícitos, assí
como birlos, pelota y axedrez y
los semejantes á éstos, y esto se
entiende jugando pocos dineros y
que se tome más por recreación
que no por vía de vicio y exercicio
continuo, de manera que por ellos
dexen de entender las gentes en
lo que les conviene, que si esto
se hace ya dexan de ser buenos y
honestos y se convierten en la
naturaleza de los que habemos
reprobado, y aun de tal manera
se podrían usar los juegos de
naipes y dados que no pudiesen
tener reprensión; pero hay pocos
que no comiencen por poco que
si tienen aparejo no vengan á
picarse y á perder ó ganar en
mucha cantidad, y por esto tengo
por mejor dexarlos del todo. Y si
queréis que concluya, todo lo
dicho es poco y casi nada, porque
son trabajos y premios y
galardones del mundo. Lo que
toca á la ánima y á la conciencia
es lo que hace al caso, y lo que
más debríamos temer y
ponérsenos delante de los ojos,
para no solamente dexar de jugar,
pero para acordarnos de jamás
tener memoria dello; y si no
hobiera prometido de no pasar
más adelante en esta materia,
todavía dixera algo que
aprovechara; pero assí quiero
dexarlo para cuando tengáis más
voluntad de oir lo que sobre esto
puedo deciros.
Bernardo.—Agora que habéis
comenzado, queremos que no
quede nada por decir, y estáis
obligado á hacerlo, pues de tan
buena gana os escuchamos y
estamos atentos al discurso de
vuestra plática.
Antonio.—Pues que assí es, yo
lo diré tan brevemente cuanto he
sido largo en lo pasado; porque
en esto no podré decir cosa
nueva, ni que dexe de estar
escrita por muchos doctores,
canonistas y legistas y teólogos
que desmenuzan y apuran esta
materia de las restituciones
declarando los decretos y leyes
en ella, altercando cuestiones y
determinando la verdad dellas,
hasta dexarlo todo en limpio; y
quien quisiese satisfacerse y verlo
todo á la clara, lea á Santo Tomás
y á Grabiel, y al Antonio,
arzobispo de Florencia, al
Cayetano, que éstos sin otros
muchos le dirán lo cierto, y
porque no dexéis de llevar alguna
cosa en suma de que podáis
aprovecharos, digo que todos les
que ganan en los juegos con
naipes ó dados falsos ó con otro
cualquier género de las
chocarrerías y traiciones que he
dicho, están obligados á
restituirlo, so pena de irse al
infierno, conforme á lo que dice
San Agustín: Non dimittitur
peccatum, nisi restituatur ablatum.
Pues lo que assí se gana, tomado
y hurtado es, siendo encubierto,
como si fuese robo manifiesto.
Anssí mesmo, todo lo que se
gana á personas que lo que
juegan no es suyo, ni pueden
disponer dello sin licencia de otra
persona, así como los criados que
juegan los dineros ó haciendas de
sus amos, los esclavos que
juegan las de sus señores, los
hijos que para esto toman las
haciendas á sus padres, los que
tienen curadores y por falta de
edad no pueden disponer de sus
haciendas, y también los que
ganan dineros á otros que saben
que los han ganado mal y están
(antes que los juegen) obligados
á la restitución dellos. Lo que se
gana á personas simples y á
enfermos necesitados, lo que se
gana atrayendo á uno por fuerza
ó por engaño ó por grandes
persuaciones á que juegue, todo
esto obliga á restitución; y en
otros muchos casos que dexo de
decir, en que hay la mesma
obligación, el cómo y cuándo y en
qué manera se haya de restituir,
déxolo para que lo veáis en los
doctores que os he dicho, y
también porque los confesores os
avisarán de ello, aunque lo mejor
sería no tener en este casso
necesidad de sus consejos.
Solamente quiero agora que
consideréis, señores, entre
vosotros, pues sois tahures y
habéis conversado y tratado con
tahures, ¿cuántos habéis visto tan
limpios y tan recatados que
tengan advertencia á estas cosas,
sino, bien ó mal, juegan con quien
quiera, trayan dineros suyos ó
sean cuyos fuesen, sean libres ó
siervos, padres ó hijos, bobos ó
sabios, los dineros que traen mal
habidos? Por cierto pocos ó
ninguno hay que dexen de hacer
á cualesquiera dineros destos, y
procurar de ganarlos de la
manera que pudieren, alegando
que no están obligados á la
especulación destas cosas, ni á
saberlas; sabiendo que la
ignorancia no excusa el pecado y
que San Pablo dice (Ad. Cor.,
xiii): Ignorans ignorabitur. Y si
queréis que os diga lo que siento
verdaderamente de los que esto
hacen, se puede presumir que no
son verdaderos cristianos, ni
sienten bien de la fe, porque más
adoran á los naipes que á Dios,
más quieren los dados qué todos
los santos, que por jurar no oyen
misa ni sermón los días de
fiestas, por el juego pierden todos
los otros oficios divinos, y se
estarán una semana sin entrar en
la iglesia; si hacen alguna oración
ó devoción es por ganar; las
cuentas que traen y lo que por
ellas rezan es echar cuentas
cómo ganarán las haciendas á
sus prójimos. Si pierden es
abominable cosa su decir mal á
Dios y blasfemar, y si lo dexan de
decir en público, es porque temen
más el castigo del cuerpo que el
del alma y el del mundo más quel
del infierno. Así que siendo
cristianos usan tan mal de la
cristiandad, que roban las
haciendas ajenas y se
aprovechan dellas, pierden el
tiempo y muchas veces pagan de
sus haciendas lo que han ganado
de las otras, de los que viven de
la manera que ellos, quedando
todos debaxo de la obligación de
restituirlas. ¿Qué diremos sin esto
de los que buscan supersticiones
y hechicerías para ganar con ellas
diciendo que tienen virtud para
ello? Y assí unos traen consigo
nóminas con nombres no
conoscidos, ó por mejor decir de
demonios, otros traen sogas de
ahorcados, otros las redecillas ó
camisas en que nacen vestidos
los niños, algunos traen
mandrágulas y otras mil
suciedades y abominaciones. Por
cierto éstos tienen en tan poco
sus ánimas, que las darán á
trueque de ganar cuatro reales
por ellas. Pues decidme, señor
Bernardo, ¿qué os parece cómo
es bueno el juego para el cuerpo
y para el alma? ¿y qué provechos
son tan grandes los que dél se
sacan? ¿No es bien dexar su
amistad y trato y conversación á
cualquier tiempo que sea, pues
que debaxo los halagos y
placeres y deleites que dél se
siguen hay tantos y tan grandes
desabrimientos, tantas afrentas y
menguas, tan terribles
desasosiegos, tanta turbación y
peligros, principalmente para la
salvación de nuestras almas?
Mirad bien en ello y consideraldo
todo, que aunque nosotros como
malos cristianos no tuviésemos
atención al daño y perjuicio de
nuestras conciencias, la
habríamos de tener á que ningún
contentamiento ni descanso de el
juego hay que después no se
vuelva en doblado trabajo y
tristeza; y nunca dió ganancia que
no se pagase con doblada
pérdida; y en fin, es siempre
mayor el dolor que se causa del
perder que la alegría que trae
consigo el ganar; y no aleguéis á
dos ó tres ó cuatro personas que
por ventura sabéis que se hayan
hecho ricos por el juego, que
éstos son como una golondrina
en el invierno, porque por ellos
veréis mill millones de gentes
perdidas y abatidas por haber
perdido cuanto tenían. Dicho os
he mi parecer y dado os he
consejo, como pienso tomarlo
para mí, y el que estoy obligado á
daros como vuestro amigo; si os
pareciere bien, seguilde, y si no
vuestro será el daño, que á mí no
me cabrá dello más de pesarme
de ver que os quedáis tan ciegos
como hasta aquí habéis estado.
Bernardo.—No penséis, señor
Antonio, que no he caído en la
cuenta de todo lo que habéis
dicho; porque vuestras palabras
me han alumbrado el juicio y
destapado los ojos del
entendimiento, que tenía ciegos, y
con firme propósito y
determinación quedo desde agora
de no jugar en mi vida, y si jugare,
á lo menos de manera que me
puedan llamar tahur por ello, que
pues decís que pasar el tiempo
entre amigos es algunas veces
lícito, no se ganando tantos
dineros que el que los perdiese
reciba daño por ello, cuando
alguna vez me desmandase será
á esto y no á más.
Antonio.—Y aun eso no ha de
ser muy continuo, porque, si
muchas veces se hiciese, de
pasatiempo se volvería en vicio, y
si pudiésedes acabar con vos de
dexar de todo punto el juego,
sería lo más seguro; pero no
quiero agora apretaros tanto que
con ello quiebre este lance que os
he armado y prisión en que de
vuestra voluntad os vais metido.
Luis.—Pues en pago de vuestra
buena intención, señor Bernardo,
y porque me prometáis de seguir
lo que agora tenéis determinado,
os quiero prestar los treinta
ducados que quedasteis
debiendo, para que, pagándolos,
cumpláis con vuestra fe y palabra.
Bernardo.—Muy gran merced es
la que me hacéis, y de los
primeros que vinieren á mí poder
seréis muy bien pagado dellos.
Antonio.—Con esto nos
podremos ir, que platicando se
nos ha passado el día y yo tengo
mucho que hacer.
Luis.—Pues comenzad á
caminar, que nosotros os
acompañaremos hasta dexaros
en vuestra posada.
Finis.
COLLOQUIO
En que se trata lo que los

médicos y boticarios están
obligados á hacer para cumplir
con sus oficios, y así mesmo
se ponen las faltas que hay en
ellos para daño de los
enfermos, con muchos avisos
necesarios y provechosos.
Divídese en dos partes: en la
primera se trata lo que toca á
los boticarios, y en la segunda
lo de los médicos.
INTERLOCUTORES
Médico, Licenciado Lerma.

Boticario, Dionisio.—Enfermo, D.
Gaspar.
Caballero, Pimentel.
Lerma.—Dios dé salud á vuestra

merced, mi señor D. Gaspar.
D. Gaspar.—Así haga á vuestra
merced para que en tiempo tan
necesario no me olvide tanto
como hoy lo ha hecho; que si no
fuera con la buena conversación
del señor Pimentel, que me ha
entretenido, muy largo se me
hubiera hecho el día, y aun con el
señor Dionisio no he holgado
poco, porque tiene gran cuidado
de visitarme, y cuando los
médicos se descuidan, es bien
que los boticarios (como uno de
sus miembros) vengan á cumplir
sus faltas con los enfermos.
Lerma.—Buena manera es essa
de reñir conmigo una falta que
hago por no poder hacer menos;
y no la hiciera sino con dexar á
vuesa merced esta mañana en
tan buena disposición, que creo
que debe estar ya sin calentura.
D. Gaspar.—Mejor viva yo que
estoy sin ella.
Lerma.—Muéstreme vuestra
merced el pulso. En verdad que
no es tanta que se pueda decir
calentura, y de aquí á mañana yo
sé cierto que no habrá ninguna.
D. Gaspar.—Menos cuenta tengo
con ella que con este dolor que
siento en el hígado, porque yo os
digo, señor licenciado, que me
atormenta tanto, que le temo, y
esto es lo principal para que yo
querría que me buscásedes
remedio.
Pimentel.—A lo que yo siento,
más debe proceder el accidente
de la calentura del mal que hay
en el hígado que no el mal ó dolor
del hígado de la calentura, y
pocas veces el señor Gaspar
estará sin ella hasta que esté
remediada la causa principal de á
donde se sigue el daño.
Lerma.—Vuestra merced dice
gran verdad, pero, según esto,
Dionisio no ha hecho el emplasto
de melliloto que yo dexé
ordenado, ni vuestra merced lo
debe tener puesto.
Dionisio.—Así es verdad.
Lerma.—¿Pues por qué no se
hizo?
Dionisio.—Porque no ha tantas
horas que vuestra merced lo
ordenó que no se pueda haber
sufrido sin él, como se han
pasado tantos días que el señor
don Gaspar lo hubiera de haber
tenido con otros beneficios que se
le pudieran haber hecho antes de
ahora.
Lerma.—¿Y qué descuidos
parece á vos que se ha tenido en
esso?
Dionisio.—Yo no he visto que
hayan precedido los remedios
universales á los particulares que
agora se hacen; pues no se han
hecho las evacuaciones conforme
á las reglas de medicina, las
cuales han de preceder á las
unciones y emplastos, según la
doctrina de Ipocras en sus
aforismos.
Lerma.—No es malo que queráis
vos haceros dotor en Medicina sin
saber letra della y que os parezca
que estoy yo obligado á sufrir
vuestra desvergüenza de
enmendarme la cura que yo hago.
¿Sabéis vos por ventura la
intención principal que yo he
llevado en ella, y si ha habido
otros accidentes más principales
y que tienen más necesidad de
remediarse?
Dionisio.—Lo que yo sé es que
no está toda la fuerza en el
emplasto para sanar el hígado.
Lerma.—Si no tuviera respeto á
estos señores que están
presentes, yo os respondiera
como vos merecíades; pero assí
no quiero deciros más de que
atendáis á hacer bien lo que toca
á vuestro oficio, y no haréis poco.
Dionisio.—Vuestra merced se ha
apasionado sin razón, y en lo que
toca á mi oficio, yo lo hago de
manera que no hay de qué
reprehenderme.
Lerma.—¿Qué podéis vos hacer
más que los otros boticarios, pues
en fin sois boticario como ellos?
Dionisio.—¿Y qué suelen hacer
los boticarios que no sea muy
bien hecho?
Lerma.—Por vuestra honra
quiero callarlo, y aun por la de los
médicos, pues lo sabemos y no lo
remediamos.
Dionisio.—Si vuestra merced lo
dixese, no faltará para ello
respuesta; pues no es justo que
en esse caso paguen justos por
pecadores.
Pimentel.—Lo que aquí se dixere
no saldrá desta puerta afuera, y
con esta condición, y con que sea
sin ningún enojo, el señor don
Gaspar y yo recebiremos muy
gran merced en que se trate algo
desta materia para satisfacerme
de algunas cosas que me han
puesto duda y sospecha de que
algunos boticarios no cumplen
con el mundo y con Dios lo que
son obligados.
Lerma.—Ningún engaño recibe
vuestra merced en esso, y plega
á Dios que no sean todos los que
esso hacen, y pues que aqui
puede pasar, menester es que
todas sean verdades las que se
dixeren.
Dionisio.—Diga vuestra merced
lo que quisiere, que ninguna pena
recebirá dello con tal que yo sea
también oído antes que la
cuestión se determine, pues estos
señores han de ser jueces della.
D. Gaspar.—Razón tiene Dionisio
en lo que pide.
Lerma.—Yo soy contento de que,
cuando sea tiempo, pueda
replicar y alegar de su derecho. Y
porque vuestras mercedes
entiendan que no me muevo sin
razón á lo que he dicho, sepan
que las condiciones que han de
tener los boticarios escriben
muchos autores, y quien
particularmente las trata, es
Saladino en la primera parte de
su obra; y porque referir todo lo
que dice sería confusión y
prolijidad, diré algunas cosas
dellas. Y lo primero es que el
boticario ha de ser de muy buen
ingenio, hombre sin vicios, sabio y
experimentado en su oficio; no ha
de ser avariento, ni deseoso de
adquirir hacienda; sobre todo ha
de ser muy fiel para que no haga
cosa contra su conciencia, ni por
su parecer, sino con consejo de
médico docto, y que en el precio
de las medicinas sea convenible.
Estas son cosas tan necesarias,
que obligan tanto al boticario á
guardarlas y cumplirlas, que no lo
haciendo, no es poco el daño ni
pocos los inconvenientes que
dello se siguen á los enfermos;
pero yo he hablado sin perjuicio
de los buenos boticarios (que son
tan pocos, que apenas se hallará
uno entre ciento), diré lo que
cerca desto hacen. Lo primero en
lo que toca á ser hombre sabio y
experimentado en su oficio no
tienen ellos toda la culpa, que la
mayor parte se puede dar á los
protomédicos porque examinan y
dan por hábiles y suficientes á
muchos que ni saben ni entienden
qué cosa son medicinas, ni tienen
experiencia dellas ni conocimiento
para alcanzar cuál es una ni cuál
es otra, sino que si van á la feria á
comprar sus drogas, no
solamente se engañan en
distinguir y apartar lo malo de lo
bueno, pero muchas veces toman
uno por otro sin conocerlo, porque
ignoran la condición y calidades
que han de tener para ser aquella
medicina que piensan; y por no se
mostrar ignorantes, quieren más
dexarse engañar de los que los
venden que tomar consejo con
quien podría desengañarlos para
que no errasen.
Pimentel.—Pues, ¿por qué los
protomédicos hacen una cosa tan
fuera de razón como essa?
Lerma.—O por no perder el
interese de los derechos que los
pagan ó porque reciben servicios
con que se obligan á hacer lo que
no deben, y sin esto aprovechan
mucho los favores de personas
señaladas ó de algunos amigos á
quien estiman en más que á las
conciencias, y así veréis que
muchos vienen examinados y con
su carta de examen muy bien
escrita y iluminada, que podrían
con más justa razón traer una
albarda que usar el oficio. Y con
poner sus boticas muy
compuestas con cajas doradas y
botes pintados, y las redomas con
unos rétulos muy grandes, á
muchas gentes hacen entender
que es oro todo lo que reluce, y
que vayan á tomar medicinas á
sus tiendas, que aprovechan más
para enfermar con ellas los sanos
que para dar salud á los
enfermos.
D. Gaspar.—En esto también me
parece que tienen la culpa los
médicos como los boticarios,
pues lo saben y lo permiten.
Lerma.—Yo no quiero excusar á
los que esso hacen.
Dionisio.—Ni podría vuesa
merced hacerlo aunque quisiese,
pero yo lo guardo todo para mi
respuesta, porque no quiero
quebrar el hilo satírico que
vuestra merced lleva tan bien
ordenado.
Lerma.—Bien es que lo hagáis
así, que también, como ya he
dicho, os oiré yo lo que en favor
vuestro y de los boticarios
alegásedes. Y tornando al
propósito, digo que es cosa recia
la desorden que en esto se tiene,
que en una cosa que va la salud y
vida de los hombres, no se ponga
mayor diligencia en conocer á los
que pueden tratar dello.
Pimentel.—¿Y qué se podría
hacer para remediarlo?
Lerma.—No dar el oficio de los
protomédicos á hombres que
hubiesen de llevar derechos ni
dineros algunos á los que
examinaren, porque así cesaría la
codicia y no los cegaría el interés
que se les sigue. Y demás desto
habíanlos de buscar personas
muy santas, temerosas de Dios y
de sus conciencias, para que no
permitiesen que ninguno tratasse
en esta arte que no la entendiese
y supiese muy bien lo que hacía.
D. Gastar.—Harto buena
gobernación sería essa, y aun
bien necesaria, si se hiciesse lo
que decís, y aun las justicias y
regimientos de los pueblos habían
de entender en remediar esta
falta, cuando saben que un
boticario no es bastante, por el
daño que dello se sigue á la

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Essentials in Modern HPLC Separations

Book • Second Edition • 2022

Front Matter, Copyright, Preface

Section I: Basic information about HPLC

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Section II: Main types of HPLC separations

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Section III: Practice of HPLC analysis

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Sample Sample Sample Core

FIGURE 1.1.1 Steps in a chemical analysis (conceptual steps in dotted frame).

Essentials in Modern HPLC Separations

I. Basic information about HPLC

I. Basic information about HPLC

I. Basic information about HPLC

I. Basic information about HPLC

Role of polarity in HPLC

I. Basic information about HPLC

I. Basic information about HPLC

I. Basic information about HPLC

I. Basic information about HPLC

I. Basic information about HPLC

I. Basic information about HPLC

I. Basic information about HPLC

I. Basic information about HPLC

chromatography and uses a resin containing macromolecules and of macromolecules from

I. Basic information about HPLC

Relation between the type of HPLC,

I. Basic information about HPLC

I. Basic information about HPLC

Sample collection Sample preparation HPLC

I. Basic information about HPLC

[8] http://www.chemaxon.com. mechanisms of liquid chromatography, Adv. Chroma-

I. Basic information about HPLC