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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
Essentials in Modern HPLC Separations | ScienceDirect https://www.sciencedirect.com/book/9780323911771/essentials-in-m...
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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tains a “dirty” matrix, having numerous other dam, 2021.
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Method in Chemical Analysis, Elsevier, Amsterdam,
the chromatographic column, must be avoided
2017.
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retention relationship, Chem. Rev. 107 (2007) [31] J. Pesek, M.T. Matyska, A comparison of two separa-
3212e3246. tion modes: HILIC and aqueous normal phase
[19] D. Casoni, J. Petre, V. David, C. Sârbu, Prediction of chromatography, LC GC N. Am. 25 (2007) 480e490.
pesticides lipophilicity from the computational molec- [32] J. Soukup, P. Jandera, Adsorption of water from
ular descriptors, J. Separ. Sci. 34 (2011) 247e254. aqueous acetonitrile on silica-based stationary phases
[20] K. Valk o, Biomimetic chromatography to accelerate in aqueous normal-phase liquid chromatography,
drug discovery: part 2, LC GC Eur. 31 (2018) 250e257. J. Chromatogr. A 1374 (2014) 102e111.
[21] K.L. Valk o, Lipophilicity and biomimetic properties [33] L. Zhang, Q. Dai, X. Qiao, C. Yu, X. Qin, H. Yan,
measured by HPLC to support drug discovery, Mixed-mode chromatographic stationary phases:
J. Pharm. Biomed. Anal. 130 (2016) 35e54. recent advancements and its applications for high-
[22] M. Chrysanthakopoulos, F. Tsopelas, A. Tsantili- performance liquid chromatography, TrAC Trends
Kakoulidou, Biomimetic chromatography: a useful Anal. Chem. 82 (2016) 143e163.
tool in the drug discovery process, Adv. Chromatogr. [34] S.N. Lanin, Y.S. Nikitin, Molecular interactions in
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[23] C. Giaginis, A. Tsantili-Kakoulidou, Quantitative 33e49.
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to simulate biological processes, Chromatographia 76 [36] G. Lunn, L.C. Hellwig, Handbook of Derivatization Re-
(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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inferred to be the universal author of all things beautiful and right,
parent of light and lord of light in this world, and the source of truth
and reason in the other: this is the first great cause which he, who
would act rationally either in public or private life, must behold.”
To the Sophist, who follows the opinion of the many instead of
regarding fixed principles of truth, he pays his respects with the
searching satire of a Carlyle.
His theology, which is a part of his philosophy, has many striking

features that have commanded the astonishment of the Christian
world. “God the Creator changes not; He deceives not.” It is wrong to
do good to friends and injure enemies, for the injury of another can
be in no case just. If you have a quarrel with any one, become
reconciled before you sleep. In heaven is the pattern of the perfect
city. All things will work together for good to the just. He advocates
the severest abstract piety that, as in the conduct of the sternest
Roman or the severest Puritan, swerves not from duty. The myth of
Er, the Armenian, reminds us in many points of the judgment day;
and his exhortation to pursue the heavenly way that it may be well
with us here and hereafter, may be our salvation if we are obedient,
is one of the most striking in the history of religious belief.
In the fifth book of the “Laws” is an exhortation to right living that
partakes of the spirit of the Christian philosophy. Every man is to
honor his own soul with an honor that regards divine good, to value
principle higher than life, to place virtue above all gold, to glory in
following the better course, to count reverence in children a greater
heritage than riches, to regard a contract as a holy thing, to avoid
excess of self-love and to adhere to the truth as the beginning of
every good. We need no further illustration of the fact that Platonism
was naturally welcomed by the early Christian Church.
The ethical ideals of Plato are the most valuable phase of his
writings. In the First Book of the “Republic,” Thrasymachus, in a
dialogue with Socrates, defines justice to be Sublime Simplicity, and
argues that the unjust are discreet and wise, as some may argue to-
day that shrewd dishonesty is commendable. The ethics of Plato is
the opposite pole of this philosophy, and as such stands for the
rational and moral order of the world. His system is not hedonistic,
but ideal. It aims at a good, but the good is attained by a life of virtue.
In a famous passage of the “Republic,” the transcendently just
man is described. He is to be clothed in justice only. Being the best
of men, he is to be esteemed the worst, and so continue to the hour
of his death. He is to be bound, scourged, and suffer every kind of
evil, and even be crucified; still he is to be just for righteousness’
sake. No wonder some Christian fathers believed this referred to
Him who was to come, as described in the celebrated chapter of
Isaiah. The best man is also the happiest, whether seen or unseen
by gods and men. In the “Crito” Socrates will not escape from prison
if it is not right, though he suffer death or any other calamity. “Virtue
is the health and beauty and well-being of the soul, and vice is the
disease and weakness and deformity of the soul.” He is a fool who
laughs at aught but folly and vice. The possession of the whole world
is of no value without the good. No pleasure except that of the wise
is quite true and pure. “Is not the noble that which subjects the beast
to the man, or rather to the god in man?” “How would a man profit if
he received gold and silver on the condition that he was to enslave
the noblest part of him to the worst?” “The Holy is loved of God
because it is Holy.” Not pleasure, but wisdom and knowledge and
right opinions and true reasonings are better, both now and forever.
The good ruler considers not his own interest, but that of the state.
The governing class are to be told that gold and silver they have
from God; the divine metal is in them.
Any one who finds in these views a doctrine of pleasure must seek
with a prejudiced eye. Plato, as usual, anticipates later ethical
discussions, and points to the fact that there is a quality in pleasure;
and quality in conduct is the very contention of absolute moralists.
He speaks of the soul whose dye of good quality is washed out by
pleasure. The attainment of genuine well-being, the development of
divine qualities within men, was the aim, and the consciousness of
this priceless possession of rational manhood was the incidental
reward. His doctrine places before men abstract ideals of Truth,
Beauty, and Goodness, which invite the better nature by their
supreme excellence.
Plato enumerates four virtues: Wisdom, Courage, Temperance,
Justice. Professor Green interprets them in modern form, and
maintains their fixed standard of excellence and universal
application. Any modern analysis of the principles of conduct which
contribute to health of soul and are favorable to success in life,
would confirm the enumeration of the Greek virtues. Professor
Green says: The Good Will is the will (1) to know what is true and to
make what is beautiful; (2) to endure pain and fear; (3) to resist the
allurements of pleasure; (4) to take for one’s self and to give to
others, not what one is inclined to, but what is due. Not only does he
enjoin the spirit of justice, but the cultivation of moral courage, and,
as contrasted with lazy ignorance, the growth in wisdom which is
realization of virtue.
Wisdom played a peculiar and important part in the Greek ethics.
Vice was ignorance, because the wise man could but live according
to his best knowledge. And the Greeks, properly interpreted, were
right. Did we see virtue in all its truth and beauty, and vice in all its
deformity, we could but choose the best. Growth in wisdom was a
gradual realization in the soul of the heavenly ideas that were the
true heritage of man, and in this development the soul was gradually
perfected. This beautiful and satisfying philosophy reappears to-day
in some of the most ennobling systems of ethics the world has
produced. It makes individual and race progress an increase in
consciousness of the knowledge of truth and virtue, a revelation of
the divine within us.
The Jewish and the Christian conception of divine law as binding
man to the performance of his moral obligations was not strongly
characteristic of the Greek mind. But responsibility, without which
conduct can have no ethical significance, was by no means foreign
to Plato’s system. In the myth of Er the soul has its choice of the lot
of life, and its condition at the end of the earthly career is a requital
for the deeds done in the body. Throughout Plato’s writings the
implications of personal merit or guilt are prominent.
It is a doctrine of virtue rather than of duty. He who sees the right
and does not do it is a fool, but that is his matter. He is not bound by
any moral law to be wise. If he is virtuous it is well; if not, so much
the worse for him. Love of God is the essential of the Christian
ethics; knowledge of the Good, of the Greek. To pursue the Good
was virtue, and virtue he sets forth in world-wide contrast with vice.
Plato’s conception of justice, or right, was so exalted that some have
thought he attained in later years an insight into the nature of
conscience, or the Moral Faculty.
The Greek idea of beauty must be touched in passing. The wise
life was a beautiful life. The Beautiful was an attribute of the Deity.
They had the love of Beauty which Goethe possessed when he had
become fascinated with the study of Greek art, and exclaimed, “The
Beautiful is greater than the Good, for it includes the Good, and adds
something to it.” Plato calls the Beautiful the splendor of the True.
The youth should learn to love beautiful forms, first a single form,
then all beautiful forms and beauty wherever found; then he will turn
to beauty of mind, of institutions and laws, and sciences, and he will
gradually draw toward the great sea of beauty, and create and
contemplate many fair thoughts, and he will become conscious of
absolute beauty, and come near to God, who is transcendent beauty
and goodness.
Plato’s philosophy makes education a process of developing the

power and knowledge latent in the mind, rather than a process of
teaching. The Socratic method of drawing out is one of time-honored
use among pedagogues. Plato defines a good education as “That
which gives to the body and to the soul all the beauty and all the
perfection of which they are capable.” The ideal aim is the
harmonious or symmetrical development of the physical, mental, and
moral powers. Physical training is for the health of the soul, as well
as for the strength and grace of the body. The training of the reason
is of first importance. The æsthetic emotions are to be cultivated as a
means of moral and religious education. Memory is little
emphasized.
The artisans and laborers were simply to learn a trade; the warrior
class were to be trained in gymnastics and music. The complete
education of the highest class, or the magistrates, was to include
music and literature, gymnastics, arithmetic, geometry and
astronomy, and finally philosophy. All this was to be supplemented
by practical acquaintance with the details of civil and military
functions.
Education is the foundation of the state, and in the “Laws” he
would make it compulsory. The women are to receive the same
training as the men. Children are to be taught to honor their parents
and respect their elders. The direction in which education starts a
man will determine his future life. In early childhood education is to
be made attractive, although to unduly honor the likings of children is
to spoil them. The tales which children are permitted to hear must be
models of virtuous thought. Harmful tales concerning the gods and
heroes are prohibited, but noble traits and deeds of endurance are to
be emphasized. Youth should imitate no baseness, but what is
temperate, holy, free, and courageous; for “imitations, beginning in
early youth, at last sink into the constitution and become a second
nature.” Children must not be frightened with ghost stories and
reference to the infernal world.
Excessive athletics makes men stupid and subject to disease. The
kinds of music employed in education must inspire courage,
reverence, freedom, and temperance. Art should present true beauty
and grace, to draw the soul of childhood into harmony with the
beauty of reason. “Rhythm and harmony find their way into the
secret places of the soul, making the soul graceful of him who is
rightly educated.” Good language and music and grace and rhythm
depend on simplicity.
Arithmetic cultivates quickness, and teaches abstract number and
necessary truth. Geometry deals with axiomatic knowledge and will
draw the soul toward truth. Astronomy compels the mind to look
upward. It is to be studied not so much for practical use, as in
navigation, but because the mind is purified and illumined thereby. In
this connection Plato maintains his position against those who carp
at the so-called useless studies.
Plato’s ideal state offends the thought of conservative men more
than all else in his writings, but it was conceived in view of the
highest ideas of virtue and justice. It was simply bad psychology. He
enumerates and describes five kinds of states and the corresponding
five types of individual character. Indeed he studies justice first in the
ideal state, and then in the individual. The three impulses of the soul
are compared with the three classes of citizens in the state, and to
each he ascribes its excellence, thus forming his list of virtues. But
we cannot dwell upon this phase of Plato’s teachings. We may,
however, refer to his caricature of extreme democracy; it has a
useful modern application.
In this state the father descends to his son and fears him, and the
son is on a level with his father and does not fear him. The alien is
equal to the citizen, and the slave to the master. The master fears
and flatters his scholars, and the scholars despise their masters. The
young man is on a level with the old, and old men, for fear of
seeming morose and authoritative, condescend to the young and are
full of pleasantry and gayety. Even the animals in the democracy
show the spirit of equality, and the horses and asses march along
the streets with all the rights and dignities of freemen, and will run at
you if you do not get out of their way, and everything is just ready to
burst with liberty. The citizens become sensitive and chafe at
authority, and cease to care for the laws. Surely the statesman can
turn to Plato for wisdom, for out of this condition grows tyranny.
And, correspondingly, the democratical young man, a kind of fin de
siècle type, is described. Insolence he terms breeding, and anarchy
liberty, and waste magnificence, and impudence courage.
No wonder Plato saw that his ideal state would not be realized
until kings became philosophers, that is to say—never. Modern
dreamers might profit by his wise predictions.
Plato’s doctrine is one of ideas and idealism as contrasted with
sensations and sensationalism. It is spiritualism as contrasted with
materialism. The higher produces the lower, instead of the lower the
higher. It is the doctrine that recognizes the rational order of the
world, the transcendent nature of conscious man, and his ethical
aim. It places ideals before man, in the attaining of which he comes
to realization of his true being. It is a doctrine of rational explanation
of man’s existence. As such it has always strongly invited the
adherence of philosophers and Christians. The founders of the
church regarded Plato as directly inspired or as having derived
inspiration from the Hebrew scriptures.
The doctrine of Universals may be taken with allowance, but we
may believe that it represents the right side of philosophical thought.
It matters not much whether we hold to the view of Plato’s ideas or
native truths of the mind developed by experience or the creative
activity of the mind in knowing the outer world or the doctrine of
participation in the divine nature and divine thought or the power to
generalize from the facts of subjective and objective nature, a power
above, and not of, material nature—all these views imply man’s
spiritual and ideal character. Behind man and behind nature is the
same reality. In some sense (not the pantheistic, as commonly
understood) both are manifestations of that reality. Hence the power
of man to know the world, because it is a rational world, and
manifestation answers to manifestation, thought to thought. He who
claims that all knowledge is founded in sensation is partly right; for to
know the outer realm is to realize the inner and to know, in part, the
truth of the Universe.
Subjective ideas, in some form, must be retained in philosophy.
Our world, as a world of evolution, is orderly and has a progressive
plan; hence, according to all human conception, is the product of
ideas worked out through what are called the laws of nature.
Men have always asked what is the use of philosophy, and to-day
they repeat the question with emphasis. We appreciate the state of
mind that rejoices in consciousness of standing on the solid earth,
the courageous patience that works out with guarded induction
scientific truth, the honesty that will not substitute hasty conjecture
for fact, and the faith that works toward results to be fully realized
only in the distant future. But many scientific men are coming to
regard biological and psychological sciences as great laboratories
for philosophy. We may believe the coming problems will be solved
by the coöperation of philosophy and science. Science studies the
objective side and philosophy the subjective side of the same reality.
Philosophy has a use as an attempt to satisfy the imperative need
of men to ask the meaning of their being. It has a use as forming a
rational hypothesis concerning a First Cause, and a Final Aim. It is a
ground of belief in ideals. All speculative philosophy has been
inspired more or less by Platonism, and has given the world the
noblest, most hopeful, useful, and influential systems of ethics.
Philosophical training gives the power to view comprehensively,
connectedly, and logically any group of facts. It contains the
presuppositions of science and of our very existence. The
investigator in the forest learns many valuable details; if he ascends
the mountains, he views the landscape as a whole, and, as it were,
finds himself. Finally philosophy represents the supreme, the
spiritual, interests of man and aims at essential truth.
Will it be relegated to the shelves of archæology? The signs of to-
day appear to answer no. In the whole history of philosophy, the
mind has never been able to rest permanently in any extreme or
one-sided position or in any position that is inadequate to explain
essential facts of existence. Hence it cannot rest permanently in
materialism. A recent writer speaks of the history of philosophy as
“preëminently a record of remarkable returns of the human intellect
to ancient follies and dreams, long since outgrown and supposed to
have been consigned to oblivion.” Well! It is strange indeed if nature
has evolved a product whose needs, instincts, and native beliefs are
a lie, a product without aim or rational ground for existence. If it is so,
then pessimism is our philosophy and annihilation our best solution
of the problem of conscious life. Most men are too respectful
believers in evolution to ascribe to nature any such satanic irony.
At any rate one likes to take an excursion in this field; he feels
benefited by the trip. Men still like to seek the great fountain head of
philosophy, and take a dip in the Castalian spring—a mental bath of
this sort is a good and useful thing. They like to sit in the shady
groves of the Academy and listen to Plato or walk with Aristotle in
the environs of the Gymnasium. The mighty minds of the past have
marked out the broad outlines of truth; it is our work to fill in, to
correct. The ethical conceptions were furnished by the ancients. The
modern world has merely made them richer in content and broader
in application. The deeper meaning of any philosophy or science is
learned by the historic method, which gives us the trend of events.
The closing words of the “Republic” are an appropriate ending to

the discussion of Plato: “And thus, Glaucon, the tale has been saved
and has not perished, and may be our salvation, if we are obedient
to the spoken word; and we shall pass safely over the river of
Forgetfulness and our soul will not be defiled. Wherefore my counsel
is that we hold fast to the heavenly way and follow after justice and
virtue always, considering that the soul is immortal and able to
endure every sort of good and every sort of evil. Thus shall we live,
dear to one another and to the gods, both while remaining here and
when, like conquerors in the games who go round to gather gifts, we
receive our reward. And it shall be well with us both in this life and in
the pilgrimage of a thousand years which we have been reciting.”
“Plato, thou reasonest well!—

Else whence this pleasing hope, this fond desire,
This longing after immortality?
’Tis the divinity that stirs within us;

’Tis heaven itself that points out an hereafter,
And intimates eternity to man.”
SECONDARY EDUCATION: A REVIEW.[1]
The manner of investigation of the Committee of Ten took a

somewhat different turn from what was anticipated when the original
report which led to the undertaking was made, but no one now
doubts the wisdom of the plan finally adopted. It would be difficult to
find groups of men in America better fitted than the members of the
conferences appointed by the Committee to discuss the specific
subjects assigned them; and their recommendations as to choice of
matter for secondary schools, the time element, place of studies in
the curriculum, and the best methods constitute a most valuable
contribution to the educational literature of the period. In the main,
they represent the best thought of practical educators.
We shall not enter into a discussion of the details of these
conference reports; each report and, in many instances, each
section of a report is in itself a large theme. The summary of results
and the recommendations of the Committee of Ten will occupy the
time allotted.
It was expected that the report as a whole would excite much
discussion and invite extensive criticism; and if no other result is
attained than the sharpening of wits in controversy, the existence of
the report has sufficient warrant.
It is impossible to say of any opinions that they are final, and of
any methods that they are the best. Some hold that the eternal
verities are to be discovered in the consciousness of the few
geniuses, and that obtaining a consensus of opinion is not the way to
reach wise conclusions. If we are Hegelian in our philosophy of
history, we shall hold to the law of development, shall believe that
each stage of thought is a necessary one, that the best light is
obtained by the historic method, and that the highest evolution of
thought is to be found in the belief and practice of the advanced
representatives of any line of investigation. The work of the
conferences was to correlate the parts of each subject by the
method of applying reason to history; it was the work of the
committee proper to correlate these results by the same method.
Whether the committee was large and varied enough to represent all
sides is to be decided by the discussions of those best fitted to form
opinions.
After a careful review of the work of our committee, I venture to

make a formal list of opinions presented, most of which, I think,
should be heartily indorsed, reserving till later the discussion of a few
of them:
1. That work in many secondary-school studies should be begun
earlier.
2. That each subject should be made to help every other, as, for
example, history should contribute to the study of English, and
natural history should be correlated with language, drawing,
literature, and geography.
3. That every subject should be taught in the same way, whether
in preparation for college or as part of a finishing course.
4. That more highly trained teachers are needed, especially for
subjects that are receiving increased attention, as the various
sciences and history.
5. That in all scientific subjects, laboratory work should be
extended and improved.
6. That for some studies special instructors should be employed to
guide the work of teachers in elementary and secondary schools.
7. That all pupils should pursue a given subject in the same way,
and to the same extent, as long as they study it at all.
8. That every study should be made a serious subject of
instruction, and should cultivate the pupil’s powers of observation,
memory, expression, and reasoning.
9. That the choice between the classical course and the Latin-
scientific course should be postponed as long as possible, until the
taste and power of the pupil have been tested, and he has been able
to determine his future aim.
10. That twenty periods per week should be adopted as the
standard, providing that five of these periods be given to unprepared
work.
11. That parallel programmes should be identical in as many of
their parts as possible.
12. That drawing should be largely employed in connection with
most of the studies.
13. The omission of industrial and commercial subjects. This is
mentioned without comment.
14. That more field work should be required for certain sciences.
15. The desirability of uniformity. Not definitely recommended in
the report.
16. That the function of the high schools should be to prepare for
the duties of life as well as to fit for college.
17. That colleges and scientific schools should accept any one of
the courses of study as preparation for admission.
18. That a good course in English should be required of all pupils
entering college.
19. That many teachers should employ various means for better
preparation, such as summer schools, special courses of instruction
given by college professors, and instruction of school
superintendents, principals of high schools, or specially equipped
teachers.
20. That the colleges should take a larger interest in secondary
and elementary schools.
21. That technological and professional schools should require for
admission a complete secondary-school education.
22. That each study pursued should be given continuous time
adequate to securing from it good results.
The points of the report which I should question are as follows:

1. That Latin should be begun much earlier than now. (This is a
conference recommendation.)
2. That English should be given as much time as Latin.
(Conference recommendation.)
3. The large number of science subjects recommended, with loss
of adequate time for each.
4. The omission of a careful analysis of the value of each subject,
absolute and relative, preparatory to tabulating courses.
5. The apparent implication that the multiplying of courses is
advisable.
6. The implications that the choice of subjects by the pupils may
be a matter of comparative indifference—the doctrine of equivalence
of studies.
7. Some parts of the model programmes made by the committee.
An examination of tabulated results of the investigations of the

conferences will show that in their opinion the following studies
should be begun below the high school:
English literature.
German or French.
Elementary algebra and concrete geometry.
Natural phenomena.
Natural history.
Biography and mythology, civil government, and Greek and
Roman history.
Physical geography.
There has been much discussion within a few years as to
improvements in elementary courses of study, with a growing
tendency toward important modifications. Rigid and mechanical
methods and an exaggerated notion of thoroughness in every detail
have often become a hindrance to the progress of the pupils in
elementary schools. The mind of the child is susceptible of a more
mature development at the age of fourteen than is usually attained.
There are numerous examples of pupils in graded schools, who, with
very limited school terms, prepare for the high school at the age of
fourteen. Under the guidance of painstaking and intelligent parents
or private tutors, children cover, in a very brief time, the studies of
the grammar school. All have noted, under favoring conditions, a
surprising development, at an early age, in understanding of history,
literature, and common phenomena, a growth far beyond that
reached at the same age in the schools. These facts simply show
the possibilities of the period of elementary education. We
understand that ultimately those best prepared to judge must
determine the modifications, if any are needed, of the elementary
courses. Some say the courses are already overcrowded, it is
impossible to add anything. Is it not true, however, that by placing
less stress upon a few things, by arousing mental activity through the
stimulus of the scientific method, and by improving the skill of the
teachers, the work suggested by these conferences may be easily
accomplished? All these experiments are already old in many
schools in the country.
Consider the logical order of studies. Each child, almost from the
dawn of consciousness, recognizes relations of number and space,
observes phenomena and draws crude inferences, records in his
mind the daily deeds of his associates, and employs language to
express his thought, often with large use of imagination. Already has
begun the spontaneous development in mathematics, science,
history, and literature. Nature points the way and we should follow
the direction. These subjects in their various forms should be
pursued from the first. Hill’s “True Order of Studies” shows that there
are some five parallel, upward-running lines representing the
divisions of knowledge, and that development may be compared to
the encircling, onward movement of a spiral, which, at each turn,
cuts off a portion of all the lines. If we accept this view, we must
grant that geometry on its concrete side belongs to the earliest
period of education; that the observation of natural phenomena with
simple inferences will be a most attractive study to the child; that the
importance of observation of objects of natural history is
foreshadowed by the spontaneous interest taken in them before the
school period; that tales of ancient heroes, and the pleasing myths of
antiquity, together with the striking characters and incidents of Greek
and Roman history, belong to the early period of historic knowledge;
that the whole world of substance and phenomena that constitutes
our environment should be the subject of study under the head of
physiography or physical geography; that the thoughts of literature,
ethical and imaginative, appeal readily to the child’s mind. We may
add that the taste of children may be early cultivated, and that the
glory which the child discovers in nature makes possible the art idea
and the religious sentiment. The reason for beginning a foreign
language early is somewhat independent, but all agree that early
study of a living language is desirable.
Should we not reconsider our analysis of the elementary courses?
Superintendents and teachers will find the necessary changes not
impossible but easy. The sum of all that is recommended for the
elementary schools by the conferences is not so formidable as at
first appears.
In the conference reports to the Committee of Ten are some views

that have a bearing upon the subject of the high-school period. The
Latin Conference hopes for a modification of the grammar-school
courses, that the high-school course may be begun earlier. The
Greek Conference voted that the average age at which pupils enter
college should be lowered. The Conference on English was of the
opinion that English work during the last two years of the grammar-
school course should be in the hands of a special teacher or
teachers. The Conference on Modern Languages holds that
whenever competent teachers can be secured the grammar school
should have an elective course in French or German. The Physics
Conference recommended that “Whenever it is possible, special
science teachers or superintendents should be appointed to instruct
teachers of elementary schools in the methods of teaching natural
phenomena.” The History Conference thought it desirable that in all
schools history should be taught by teachers who have a fondness
for historical studies and have paid special attention to effective
methods of imparting instruction. One member of the conference
was almost ready to advise omitting history from school programmes
because of so much rote, text-book teaching.
These opinions are additional evidence of need of modifications in
grammar-school work, and some think that ultimately the best
solution will be found in extending the high-school period downward
to include part of the elementary period.
It was agreed in the Committee of Ten that their task would be less
difficult did the high-school period begin, say two years earlier; and
the reason why the recommendation of the conferences, that certain
studies be introduced below the high school, was viewed with
suspicion was the impossibility, with the present organization of the
schools, of securing good instruction in these studies.
The following view of the high-school period is expressed by a
prominent high-school principal: “My opinion is that it would be much
better for our boys and girls to begin their preparation for college at
least two years earlier than they now do. If our high schools could
receive the pupils at eleven or twelve, instead of fourteen,
preparation for college would be completed at sixteen instead of
eighteen, as is now generally the case.”
The custom in European countries supports the view that high-
school methods should reach down into the grades. In Prussia only
three years of elementary work precede the gymnasium, and the
pupil can enter the gymnasium at the age of nine. The gymnasium
itself covers a period of nine years, extending five years below the
period of our high schools. Examining the course of the Prussian
gymnasium, we find in the first five years, or before the age of
fourteen, Latin, Greek, French, history, geometry, natural history; and
it is conceded by many educators that more is attained by the age of
eighteen in Germany than in this country; that at the age of fourteen
in Germany the development of the pupil is more mature, and that in
essential features of education he has made more desirable
progress.
If our high schools should be made equivalent in length and rank
to the Prussian gymnasium, the change would involve the entire
reconstruction of our school system, from the primary school to the
end of the university. The high schools would become colleges, and
the colleges would become high schools, and the graduates from
them would enter the university prepared to take up professional or
other special university work. That there are many leading educators
who advocate these changes for the universities is well known, and
there are some strong tendencies toward the German system. On
the other hand, many deplore the possibility of losing the American
college, which is an institution somewhat peculiar to this country.
They think that its broad, general education and superior culture are
worth retaining, and that specialization should begin at a late period.
One significant fact stares us in the face, namely, that the average
American boy no longer will spend four years beyond the high school
in general education, and then pass four years more at the
professional school or three years in the graduate course.
Somewhere the work must be shortened, in either the elementary
school, the high school, or the college.
The whole subject is of great interest and importance, but at the
present stage of inquiry no definite conclusions can be reached.
The relation of the mind to a study is determined by the nature of
the mind and the nature of the study, and there seems to be no
reason in psychology why a college-preparatory subject should be
taught differently to one fitting for the duties of life. Besides, it is
economy to make identical the work of different courses, as far as
possible. There was perfect unanimity in the opinion that the same
studies should be pursued by all in the same way, as far as taken.
Every one knows that many teachers are unskilled to present in
the elementary schools the beginnings of geometry, science, history,
or literature, and that the failures in this work are due to the
mechanical efforts of those who have had no higher or special
training. The demands of present methods are imperative for
improved power in instruction. Science is not well taught in all
schools. There is a school which teaches biology from a manual
without specimen, microscope, or illustrations. It was a humiliating
confession of the committee that the classical course is superior, for
the reason that it is difficult to find enough instructors competent to
teach modern subjects by modern methods.
A very important point, recognized by the committee, is the
advantage of postponing as long as possible the necessity of making
a final choice of courses. In this country we have no fixed conditions
of rank, and the poor man’s son has the same privileges as the sons
of position and wealth. Hence, the station in life is not determined by
the differentiation in courses at an early period. Very few parents
decide upon the final character of the child’s instruction much before
the beginning of the college period.
For these reasons many would not agree with the conference
recommendation to begin Latin at an earlier period. It would not be
economy; there is enough else that belongs to the elementary stage
of education, and no plan is feasible that is founded upon the foreign
view of caste and fixed condition in life.
Uniformity in requirements for admission to college was the
subject of the report that finally led to this investigation. Although
uniformity is not prominently urged in the report of the Committee of
Ten, doubtless the logical outcome of the latter report will be a
tendency toward some kind of uniformity. There is a vigorous conflict
of opinion to-day as to nationalism and individualism, with a strong
tendency, especially in education, toward individualism. In the
opinion of many there exists a harmful slavery of the high and
preparatory schools to the erratic and varied demands of different
colleges, and also a slavery to ignorance and caprice in some
schools themselves, which would be removed by a general
agreement to uniformity. Men are not enslaved, but are
emancipated, by organization, and freedom of the individual is found
in the good order of society and government. In a facetious criticism
of the committee’s report, arguing for extreme individualism in choice
of studies, appears the following query: “Please tell us if you and
your colleagues on the conference considered any methods for the
encouragement of cranks?” No; for the encouragement neither of
cranks, nor of crankiness, but for the encouragement of the best kind
of rational education. While there are a few wise, independent
investigators who need no enforced uniformity, and will not be bound
by the recommendations of others, many of the schools are largely
imitators, or, worse, are working independently with limited insight,
and these schools would be vastly improved by adopting courses
and methods growing from a consensus of the best opinions of the
country. The lowest would thereby tend to rise to the highest, and
from that plane a new advance could be made. Meantime the
original thinkers would be free to push forward toward higher results,
to be generally adopted later. Through contact of various ideas some
principles are settled, and the world is free to move on toward fresh
discovery.
The selection of studies is to be determined largely by the nature
of the mind and by the universal character of natural and civil
environments, and this fact points toward the possibility of uniformity.
The period of secondary education is not the period for specializing,
and even if it were, there should be some uniformity in differentiation.
In the United States there is, broadly speaking, uniformity of
tradition, of government, of civilization, and the educated youth of
San Francisco bears about the same relation to the world as the
educated youth of Boston; hence, so far as elementary and
secondary education is pursued, there is no reason why it should not
be substantially the same in various schools—not in details
belonging to the individual teacher, but in paper requirements and
important features of methods.
Nothing in the whole report is more important than the proposed

closer connection between high schools and colleges, and this is
clearly and forcibly urged. Whatever course of study properly
belongs to a secondary school is also a good preparation for higher
education, else either secondary or higher education is seriously in
error. Whenever a youth decides to take a college course, he should
find himself on the road toward it. No one can doubt that in the
coming years pupils having pursued properly arranged high-school
courses must be admitted to corresponding courses in higher
education. The divorcement between higher education and all lower
grade work, except the classical, has been a fatal defect in the past.
The entire course of education should be a practical interest of
college professors, and there should be a hearty coöperation
between them and school superintendents and principals in
considering all educational problems.
It is a fact of significance that a committee, on which some leading
institutions are represented, urges the professional schools of the
country to place their standard of admission as high as that of the
colleges; and we hope that aid will thus be given the institutions
endeavoring to raise the requirements of law, medical, and divinity
schools.
The reports of most of the conferences asked for continuous and
adequate work for each subject, that it might become a source of
discipline and of valuable insight. No doubt part of the work in high
schools is too brief and fragmentary to gain from it the best results.

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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