28

chapter

High-Performance Liquid
Chromatography

Bradley L. Reuhs∗ and Mary Ann Rounds

Department of Food Science, Purdue University,
West Lafayette, IN 47907-2009, USA
[email protected]

28.1 Introduction 501 28.2.3.2 HPLC Column Packing

28.2 Components of an HPLC System 501 Materials 503
28.2.1 Pump 501 28.2.3.2.1 General
28.2.2 Injector 502 Requirements 503
28.2.3 Column 503 28.2.3.2.2 Silica-Based Column
28.2.3.1 Column Hardware 503 Packings 504
28.2.3.1.1 Precolumns 503 28.2.3.2.3 Porous Polymeric
28.2.3.1.2 Analytical Column
Columns 503 Packings 504

S.S. Nielsen, Food Analysis, Food Science Texts Series, DOI 10.1007/978-1-4419-1478-1_28, 499
°c Springer Science+Business Media, LLC 2010
500 Part V • Chromatography

28.2.4 Detector 505 28.3.3 Ion Exchange 509

28.2.4.1 UV-Vis Absorption 28.3.3.1 Stationary and Mobile
Detectors 505 Phases 509
28.2.4.2 Fluorescence Detectors 505 28.3.3.2 Applications of Ion-Exchange
28.2.4.3 Refractive Index HPLC 509
Detectors 505 28.3.3.2.1 Ion Chromatogra-
28.2.4.4 Electrochemical Detectors 505 phy 509
28.2.4.5 Other HPLC Detectors 506 28.3.3.2.2 Ion Exchange
28.2.4.6 Coupled Analytical Chromatography of
Techniques 506 Carbohydrates and
28.2.4.7 Chemical Reactions 506 Proteins 510
28.2.5 Data Station Systems 507 28.3.4 Size Exclusion 510
28.3 Applications in HPLC 507 28.3.4.1 Column Packings and Mobile
28.3.1 Normal Phase 507 Phases 510
28.3.1.1 Stationary and Mobile 28.3.4.2 Applications of High
Phases 507 Performance SEC 511
28.3.1.2 Applications of Normal-Phase 28.3.5 Affinity 511
HPLC 508 28.4 Summary 511
28.3.2 Reversed Phase 508 28.5 Study Questions 512
28.3.2.1 Stationary and Mobile 28.6 Acknowledgments 512
Phases 508 28.7 References 512
28.3.2.2 Applications of Reversed-Phase
HPLC 508
Chapter 28 • High-Performance Liquid Chromatography 501

28.1 INTRODUCTION analysis of separated components is needed. Con-

necting tubing, tube fittings, and the materials out
High-performance liquid chromatography (HPLC) of which components are constructed also influence
developed during the 1960s as a direct offshoot system performance and lifetime. References (1) and
of classic column liquid chromatography through (6–10, 15) include detailed discussions of HPLC equip-
improvements in the technology of columns and ment, with the book by Bidlingmeyer (1) especially
instrumental components (pumps, injection valves, appropriate for beginners. The unique organization of
and detectors). Originally, HPLC was the acronym reference (8) is intended for those who may need to
for high-pressure liquid chromatography, reflecting the learn chromatography quickly in an industrial envi-
high operating pressures generated by early columns. ronment. Two useful books on HPLC troubleshooting
By the late 1970s, however, high-performance liq- are those written by Gertz (11) and Dolan and Sny-
uid chromatography had become the preferred term, der (12). In addition, much information on HPLC
emphasizing the effective separations achieved. In equipment, hardware, and troubleshooting hints may
fact, newer columns and packing materials offer high be found in publications such as LC·MS, American
performance at moderate pressure (although still high Laboratory, Chemical & Engineering News, and simi-
pressure relative to gravity-flow liquid chromatog- lar periodicals. Manufacturers are also a source of
raphy). HPLC can be applied to the analysis of any practical information on HPLC instrumentation and
compound with solubility in a liquid that can be columns/stationary phase material.
used as the mobile phase. Although most frequently
employed as an analytical technique, HPLC also may
be used in the preparative mode. There are many
28.2.1 Pump
advantages of HPLC over traditional low pressure The HPLC pump delivers the mobile phase through the
column liquid chromatography: system, typically at a flow rate of 0.4–1 ml/min, in a
controlled, accurate, and precise manner. The majority
1. Speed (many analyses can be accomplished in
of pumps currently used in HPLC (>90%) are recip-
30 min or less)
rocating, piston-type pumps. The dual piston pump
2. A wide variety of stationary phases
systems with ball check valves are the most efficient
3. Improved resolution
pumps available. One disadvantage of reciprocating
4. Greater sensitivity (various detectors can be
pumps is that they produce a pulsating flow, requir-
employed)
ing the addition of pulse dampers to suppress fluctu-
5. Easy sample recovery (less eluent volume to
ations. A mechanical pulse damper or dampener con-
remove)
sists of a device (such as a deformable metal compo-
Application of HPLC to the analysis of food began nent or tubing filled with compressible liquid) that can
in the late 1960s, and its use increased with the devel- change its volume in response to changes in pressure.
opment of column packing materials that would sep- Gradient elution systems for HPLC are used to
arate sugars. Using HPLC to analyze sugars was jus- vary the mobile phase concentration during the run,
tified economically as a result of sugar price increases by mixing mobile phase from two or more reservoirs.
in the mid 1970s, which motivated soft drink manufac- This is accomplished with low-pressure mixing, in
turers to substitute high-fructose corn syrup for sugar. which mobile phase components are mixed before
Monitoring sweetener content by HPLC assured a entering the high-pressure pump, or high-pressure
good quality product. Other early food applications mixing, in which two or more independent, pro-
included the analysis of pesticide residues in fruits grammable pumps are used. For low-pressure gra-
and vegetables, organic acids, lipids, amino acids, tox- dient systems, a computer-controlled proportioning
ins (such as aflatoxins in peanuts), and vitamins (1). valve, followed by a mixing chamber at the inlet to
HPLC continues to be applied to these, and many the pumps is used, which results in extremely accu-
more, food-related analyses today (2–5). rate and reproducible gradients. Gradient HPLC is
extremely important for the effective elution of all
components of a sample and for optimal resolution.
28.2 COMPONENTS OF AN HPLC SYSTEM It is routinely applied to all modes of HPLC except
size-exclusion chromatography.
A schematic diagram of a basic HPLC system is shown Many commercially available HPLC pumping sys-
in Fig. 28-1. The main components of this system – tems and connecting lines are made of grade ANSI 316
pump, injector, column, detector, and data sys- stainless steel, which can withstand the pressures gen-
tem – are discussed briefly in the sections below. erated. Also it is resistant to corrosion by oxidizing
Also important are the mobile phase (eluent) reser- agents, acids, bases, and organic solvents, although
voirs, and a fraction collector, which is used if further mineral acids and halide ions do attack stainless
502 Part V • Chromatography

Schematic representation of a system for high-performance liquid chromatography (not drawn to scale).
28-1 Column(s) and detector may be thermostatted, as indicated by the dashed line, for operation at elevated
figure temperature.

steel. In other systems, all components that come into

contact with the eluent are made of sturdy, inert poly-
mers, and even employ sapphire pistons, which are
resistant to extreme pH and high salt concentration.
The latter systems can be used for all applications
except normal phase, which uses organic solvents as
the mobile phase. The polymer-based systems have
facilitated a wider application of ion exchange HPLC.
All HPLC pumps contain moving parts such as
check valves and pistons, and are quite sensitive
to dust and particulate matter in the liquid being
pumped. Therefore, it is advisable to filter the mobile
phase using 0.45 or 0.22-µm filters prior to use.
Degassing HPLC eluents, by the application of a vac-
uum or by sparging with helium, also is recommended
to prevent the problems caused by air bubbles in a
pump or detector.

28.2.2 Injector
The role of the injector is to place the sample into the
flowing mobile phase for introduction onto the col-
umn. Virtually all HPLC systems use valve injectors,
which separate sample introduction from the high-
pressure eluent system. With the injection valve in the
LOAD position (Fig. 28-2a), the sample is loaded into
an external, fixed-volume loop using a syringe. Elu-
ent, meanwhile, flows directly from the pump to the
column at high pressure. When the valve is rotated Valve-type injector. The valve allows the sam-
to the INJECT position (Fig. 28-2b), the loop becomes 28-2 ple loop to be (a) isolated from the pump elu-
figure
part of the eluent flow stream and sample is carried ent stream (LOAD position) or (b) positioned
onto the column. Such injectors are generally trouble in it (INJECT position). [from (9), used with
permission.]
free and afford good precision.
Chapter 28 • High-Performance Liquid Chromatography 503

Changing the loop allows different volumes to 28.2.3.1.2 Analytical Columns The most commonly
be injected. Although injection volumes of 10–100 µl used analytical HPLC columns are 10, 15, or 25-cm
are typical, both larger (e.g., 1–10 ml) and smaller long with an internal diameter of 4.6 or 5 mm (9).
(e.g., ≤2 µl) sample volumes can be loaded by utiliz- Short (3 cm) columns, packed with ≤3 µm particles,
ing special hardware. An important advantage of the are gaining popularity for fast separations; for exam-
loop valve design is that it is readily adapted to auto- ple, in method development or process monitoring. In
matic operation. Thus, automated sample injectors, or recent years, the use of columns with smaller inter-
autosamplers, may be used to store and inject large nal diameters (<0.5–2.0 mm), including wall-coated
numbers of samples. Samples are placed in uniform- capillary columns, has increased. The advantages of
size vials, sealed with a septum, and held in a (pos- using smaller diameter columns include a decreased
sibly refrigerated) tray. A computer actuated needle consumption of mobile phase, an increased peak con-
penetrates the septum to withdraw solution from the centration, increased resolution, and the ability to
vial, and a mechanically or pneumatically operated couple HPLC with mass spectrometry (MS) (13).
valve introduces it onto the column. Autosamplers Various names have been used for the reduced-
can reduce the tedium and labor costs associated with volume columns. Dorsey et al. (14) refer to columns
routine HPLC analyses and improve assay precision. with internal diameters of 0.5–2.0 mm as microbore,
However, because samples may remain unattended while packed or open tubular columns having inter-
for 12–24 h prior to automatic injection, sample stabil- nal diameters of <0.5 mm are termed microcolumns
ity is a limiting factor for using this accessory. or capillary columns (a capillary column is a narrow-
bore open tubular column, in which the inner surface
28.2.3 Column is coated with a thin layer of stationary phase). In
the case of the packed columns, the microbore or
28.2.3.1 Column Hardware microcolumns contain very small particle size pack-
An HPLC column is usually constructed of stainless ing material. Because of the extremely high operating
steel tubing with terminators that allow it to be con- pressures of these systems, they are often referred
nected between the injector and detector of the system to as ultra-HPLC (UHPLC). To achieve good perfor-
(Fig. 28-1). Columns also are made from glass, fused mance from microcolumns, it is essential to have an
silica, titanium, and polyether ether ketone (PEEK) HPLC system with very low dead-volume, so that
resin; the PEEK columns are essential for the high peak broadening outside the column does not destroy
pH, high salt concentrations necessary for the power- resolution achieved within the column. Pumps, injec-
ful ion exchange HPLC systems. Many types and sizes tors, and other hardware designed specifically for use
of columns are commercially available, ranging from with these columns are available from commercial
5 cm × 50 cm (or larger) preparative columns down to suppliers.
wall-coated capillary columns.
28.2.3.2 HPLC Column Packing Materials
28.2.3.1.1 Precolumns Auxiliary columns that pre-
cede the analytical HPLC column are termed pre- The development of a wide variety of column packing
columns. Short (≤5 cm) expendable columns, called materials has contributed substantially to the success
guard columns, often are used to protect the analytical and widespread use of HPLC.
column from strongly adsorbed sample components.
A guard column (or cartridge) is installed between the
injector and analytical column via short lengths of cap- 28.2.3.2.1 General Requirements A packing mate-
illary tubing (or a cartridge holder). They may be filled rial serves, first of all, to form the chromatographic
with either pellicular media (see Sect. 28.2.3.2.2) of the bed; however, in most modes of chromatography the
same bonded phase as the analytical column, or with column packing material serves as both support and
microparticulate (≤10 µm) packing material identi- the stationary phase. Requirements for HPLC column
cal to that of the analytical column. Microparticulate packing materials are good chemical stability, suffi-
guard columns are usually purchased as prepacked, cient mechanical strength to withstand pressure gener-
disposable inserts for use in a special holder, and ated during use, and the availability of a well-defined
cost much less than replacing an analytical column. particle size, with a narrow particle size distribution
A guard column (or cartridge) should be repacked or (10). Two materials that meet the above criteria are
replaced before its binding capacity is exceeded and porous silica and synthetic organic resins (see Sects.
contaminants pollute the analytical column. 28.2.3.2.2 and 28.2.3.2.3, respectively).
504 Part V • Chromatography

28.2.3.2.2 Silica-Based Column Packings Porous skeleton slowly dissolves in aqueous solutions, and
silica meets the above criteria quite well and can be the rate of this process becomes prohibitive at pH<2
prepared in a wide range of particle and pore sizes, and >8.
with a narrow particle size distribution. Both par- A pellicular packing material (Fig. 28-3b) is made
ticle size and pore diameter are important: Small by depositing a thin layer or coating onto the surface
particles reduce the distance a solute must travel of an inert, usually nonporous, microparticulate core.
between stationary and mobile phases, which facil- Core material may be either inorganic, such as silica, or
itates equilibration and results in good column effi- organic, such as poly(styrene-divinylbenzene) or latex.
ciencies (Chap. 27, Sect. 27.5.2.2.2). However, small Functional groups, such as ion-exchange sites, are then
particles also yield greater flow resistance and higher present at the surface only. The rigid core ensures good
pressure at equivalent flow rates. Spherical particles physical strength, whereas the thin stationary phase
of 3, 5, or 10-µm diameter are utilized in analytical provides for rapid mass transfer and favorable column
columns. One-half or more of the volume of porous efficiency.
silica consists of the pores (10). Use of the smallest
possible pore diameter will maximize surface area and 28.2.3.2.3 Porous Polymeric Column Packings Syn-
sample capacity, which is the amount of sample that thetic organic resins offer the advantages of good
can be separated on a given column. Packing materi- chemical stability and the possibility to vary interac-
als with a pore diameter of 50–100 Å and surface area tive properties through direct chemical modification.
of 200–400 m2 /g are used for low-molecular-weight Two major categories of porous polymeric packing
(<500 Da) solutes. For increasingly larger molecules, materials exist.
such as proteins and polysaccharides, it is necessary to Microporous or gel-type resins (Fig. 28-3c) are
use wider pore materials (pore diameter ≥300 Å), so comprised of crosslinked copolymers in which the
that internal surface is accessible to the solute (10). apparent porosity, evident only when the gel is in its
Bonded phases (Fig. 28-3a) are made by cova- swollen state, is determined by the degree of crosslink-
lently bonding hydrocarbon moieties to −OH groups ing. These gel-type packings undergo swelling and
(silanols) on the surface of silica particles (10, 16). contraction with changes in the chromatographic
Often, the silica is reacted with an organochlorosilane: mobile phase. Microporous polymers of less than ca.
8% crosslinking are not sufficiently rigid for HPLC use.
R1
Macroporous resins are highly crosslinked (e.g.,
R1
\ | \ | ≥50%) and consist of a network of microspheric
— Si — OH + C1 —Si—R3 —Si— O — Si—R3 + HC1 gel beads joined together to form a larger bead
/ | / |
R2 R2 (Fig. 28-3d). Large, permanent pores, ranging from
100 to 4000 Å or more in diameter, and large surface
[1] areas (≥100 m2 /g) are the result of interstitial spaces
between the microbeads (16). Rigid microparticulate
Substituents R1 and R2 may be halides or methyl poly(styrene-divinylbenzene) packing materials of the
groups. The nature of R3 determines whether the macroporous type are popular for HPLC use. They are
resulting bonded phase will exhibit normal-phase, stable from pH 1 to 14 and are available in a variety
reversed-phase, or ion-exchange chromatographic of particle and pore sizes. These resins can be used in
behavior. The main disadvantage of silica and silica- unmodified form for reversed-phase chromatography
based bonded-phase column packings is that the silica or functionalized for use in other HPLC modes.

Some types of packing materials utilized in HPLC. (a) Bonded-phase silica; (b) pellicular packing; (c) microporous
28-3 polymeric resin; (d) macroporous polymeric resin. [Adapted from (16), p. 621, by courtesy of Marcel Dekker, Inc.]
figure
Chapter 28 • High-Performance Liquid Chromatography 505

28.2.4 Detector Diode array spectrophotometric detectors can

provide much more information about sample compo-
A detector translates sample concentration changes in
sition than is possible with monochromatic detection.
the HPLC column effluent into electrical signals. Spec-
In this instrument, all the light from a deuterium
trochemical, electrochemical, or other properties of
lamp is spread out into a spectrum that falls across an
solutes may be measured by a variety of instruments,
array of photodiodes mounted on a silicon chip. These
each of which has advantages and disadvantages. The
are read almost simultaneously by a microprocessor
choice of which to use depends on solute type and
to provide the full absorption spectrum from 200 to
concentration, and on detector sensitivity, linear range,
700 nm every 0.1 s, which may enable the components
and compatibility with the solvent and elution mode
of a mixture to be identified. Although considerably
to be used. Cost also may influence detector selection.
more expensive than variable-wavelength detectors,
One common feature of most HPLC detectors is the
they are useful in method development and in routine
presence of a flow cell, through which the eluent flows
analyses in which additional evidence of peak identity,
as it is analyzed by the detector system. These flow
without further analysis, is needed.
cells are often delicate and easily polluted or damaged,
so care must be taken when handling them.
The most widely used HPLC detectors are based 28.2.4.2 Fluorescence Detectors
on ultraviolet-visible (UV-Vis) and fluorescence spec-
trophotometry, refractive index determination, and Some organic compounds can re-emit a portion of
electrochemical analysis (see Chap. 22 for detailed absorbed UV-Vis radiation at a longer wavelength
discussion of UV-Vis and fluorescence spectrophotom- (lower energy). This is known as fluorescence, and
etry). Many other methods, such as light scattering or measurement of the emitted light provides another
mass spectrometry, also can be applied to the detec- useful detection method. Fluorescence detection is
tion of analytes in HPLC eluents. More than one type both selective and very sensitive, providing up to
of HPLC detector may be used in series, to provide 1000-fold lower detection limits than for the same
increased specificity and sensitivity for multiple types compound in absorbance spectrophotometry (10).
of analytes. In one food-related application, a multi- Although relatively few compounds are inherently flu-
detector HPLC system equipped with a diode array orescent, analytes often are converted into fluorescent
absorption detector coupled to fluorescence and elec- derivatives (see Sect. 28.2.4.7). Ideal for trace analysis,
trochemical detectors was used to monitor a wide fluorescence detection has been used for the determi-
variety of Maillard reaction products (2). nation of various vitamins in foods and supplements,
monitoring aflatoxins in stored cereal products, and
the detection of aromatic hydrocarbons in wastewater.

28.2.4.1 UV-Vis Absorption Detectors

28.2.4.3 Refractive Index Detectors
Many HPLC analyses are carried out using a UV-Vis
absorption detector, which can measure the absorp- Refractive index (RI) detectors measure change in the
tion of radiation by chromophore-containing com- RI of the mobile phase due to dissolved analytes,
pounds. The three main types of UV-Vis absorption which provides a nearly universal method of detec-
detectors are fixed wavelength, variable-wavelength, tion. However, because a bulk property of the eluent
and diode array spectrophotometers (9). As its name is being measured, RI detectors are less sensitive than
implies, the simplest design operates at a single, fixed other types. Another disadvantage is that they cannot
wavelength. A filter is used to isolate a single emission be used with gradient elution, as any change in elu-
line (e.g., at 254 nm) from a source such as a mer- ent composition will alter its RI, thereby changing the
cury lamp. This type of detector is easy to operate and baseline signal. RI detectors are widely used for ana-
inexpensive, but of limited utility. lytes that do not contain UV-absorbing chromophores,
The most popular general purpose HPLC detec- such as carbohydrates and lipids, when the analytes
tor today is the variable-wavelength detector in which are present at relatively high concentration.
deuterium and tungsten lamps serve as sources of
ultraviolet and visible radiation, respectively. Wave-
28.2.4.4 Electrochemical Detectors
length selection is provided by a monochromator, a
device that acts somewhat like a prism to deflect light. Electroanalytical methods used for HPLC detec-
An exit slit in the monochromator allows light from tion are based either on electrochemical oxidation–
a limited range of wavelengths to pass through, and reduction of the analyte or on changes in conductivity
rotating the monochromator allows one to change the of the eluent. Amperometric detectors measure the
operating wavelength. change in current as the analyte is oxidized or reduced
506 Part V • Chromatography

by the application of voltage across electrodes of the drugs. Decay of a radioactive nucleus leads to exci-
flow cell. This method is highly selective (nonreac- tation of a scintillator, which subsequently loses
tive compounds give no response) and very sensitive. its excess energy by photon emission. Photons are
A major application of electrochemical detection has counted by a photomultiplier tube and the number
been for the routine determination of catecholamines, of counts per second is proportional to radiolabeled
which are phenolic compounds of clinical importance analyte (9).
that are present in blood and tissues at very low lev- A chemiluminescent nitrogen detector (CLND)
els. The development of a triple-pulsed amperometric allows nitrogen-containing compounds, such as amino
detector, which overcame the problem of electrode acids, to be detected without using chemical derivati-
poisoning (accumulation of oxidized product on the zation (Sect. 28.2.4.7). This nitrogen-specific detection
electrode surface), has allowed electrochemical detec- system has been used to quantify caffeine in coffee and
tion to be applied to the analysis of carbohydrates soft drink beverages, and to analyze capsaicin in hot
(see Sect. 28.3.3.2.2). Pulsed electrochemical detection peppers (2).
also has excellent sensitivity for the quantification of
flavor-active alcohols, particularly terpenols (2).
Analytes that are ionized and carry a charge can 28.2.4.6 Coupled Analytical Techniques
be detected by measuring the change in eluent conduc-
To obtain more information about the analyte(s), elu-
tivity between two electrodes. Conductivity detection
ent from an HPLC system can be passed on to a second
has been used mainly to detect inorganic anions and
analytical instrument, such as infrared (IR), nuclear
cations and organic acids upon elution from weak
magnetic resonance (NMR), or MS [see Chaps. 23, 25,
ion-exchange columns. Its principal application has
and 26, respectively, or reference (6)]. The coupling of
been the basis of ion chromatography (Sect. 28.3.3.2.1).
spectrometers with liquid chromatography (LC) was
An excellent overview of electrochemical detection is
initially slow to gain application, due to many prac-
provided by Swedesh (8).
tical problems. For example, in the case of HPLC
with mass spectrometric detection (LC–MS), the liq-
uid mobile phase affected the vacuum in the MS. This
problem was addressed by the development of com-
28.2.4.5 Other HPLC Detectors mercial interfaces that allow solvent to be evaporated
so that only analyte is carried to the spectrometer. Two
Unfortunately, there is no truly universal HPLC detec-
commonly used interface techniques are discussed in
tor with high sensitivity. Thus, there have been many
detail by Harris (6). The use of microbore or capillary
attempts to find new principles that could lead to
HPLC columns with a low flow volume also facilitates
improved instrumentation. One interesting concept is
direct coupling of the two instruments (13). LC–MS
the evaporative light scattering detector. The mobile
systems continue to improve, and the applications
phase is sprayed into a heated air stream, evapo-
are expanding to nearly every class of relatively low
rating volatile solvents and leaving nonvolatile ana-
molecular weight compounds, including bioactives
lytes as aerosols. These droplets or particles can be
and contaminants.
detected because they will scatter a beam of light
(8). HPLC with light scattering detection has been
applied to the analysis of wheat flour lipids. Also,
28.2.4.7 Chemical Reactions
light scattering detectors are quite useful for the char-
acterization of polymers by size-exclusion chromatog- Detection sensitivity or specificity may sometimes be
raphy. Improvements in laser applications brought enhanced by converting the analyte to a chemical
about the development of low-angle laser light scat- derivative with different or additional characteristics.
tering (LALLS) and multi-angle laser light scattering An appropriate reagent can be added to the sample
(MALLS) detectors. With these detectors, there is no prior to injection (i.e., precolumn derivatization) or
need to evaporate the mobile phase, as the laser beam combined with column effluent before it enters the
is directed at the flow cell, and scattered laser light detector (i.e., postcolumn derivatization). Automated
is then monitored by photo detectors set at specific amino acid analyzers utilize postcolumn derivatiza-
angles to the cell. In MALLS there may be as many tion, usually with ninhydrin, for reliable and repro-
as 18 different photo detectors at discrete angles, each ducible analysis of amino acids. Precolumn derivati-
continuously collecting and analyzing the scattered zation of amino acids with o-phthalaldehyde or similar
light; from this data, the computer can determine the reagents permits highly sensitive HPLC determination
molecular weight of the eluting sample. of amino acids using fluorescence detection (Chap. 15,
Radioactive detectors are widely used for phar- Sect. 15.3.1.2). In addition, fractions may be collected
macokinetic and metabolism studies with radiolabeled after passing through the detector and aliquots of each
Chapter 28 • High-Performance Liquid Chromatography 507

fraction analyzed by various means, including chem- developing the gradient via control of the proportion-
ical/colorimetric assays, such as the Lowry protein ing pump systems. When combined with an autosam-
assay (Chap. 9, Sect. 9.2.5) or a total carbohydrate pler, the data station can carry out the entire operation,
assay (Chap. 10, Sect. 10.3.2). The results can then on hundreds of samples, in the absence of an operator,
be plotted and overlaid with the detector plot, yield- and, with networking, deliver the analysis file/report
ing very important information about the compounds to an office computer.
eluting in various peaks.

28.3 APPLICATIONS IN HPLC

28.2.5 Data Station Systems
The basic physicochemical principles underlying all
A detector provides an electronic signal related to the
liquid chromatographic separations – adsorption, par-
composition of the HPLC column effluent. It is the job
tition, ion exchange, size exclusion, and affinity –
of the last element in the chain of HPLC instrumen-
are discussed in Chap. 27, and details will not be
tation to display the chromatogram, and integrate the
repeated here. The number of separation modes uti-
peak areas. Data stations and software packages are
lized in HPLC, however, is greater than that avail-
nearly ubiquitous with modern HPLC, and all come
able in classic chromatography. Examples of HPLC
with very powerful tools for sample identification
applications in food analysis are given in Table 28-1.
and quantitation. As an HPLC analysis progresses,
This is attributable to the success of bonded phases,
the data from the HPLC detector(s) are digitized and
initially developed to facilitate liquid–liquid parti-
saved to the hard drive of a dedicated computer. The
tion chromatography (Chap. 27, Sect. 27.4.2). In fact,
operator can then manipulate the data, by assigning
reversed-phase chromatography is the most widely
and integrating the peaks, for example, and then print
used separation mode in modern HPLC.
out plots and tables for further assessment. Impor-
tantly, the software programs can be set up prior to the
analysis to perform nearly all these functions, without 28.3.1 Normal Phase
further input from the operator. For example, retention
28.3.1.1 Stationary and Mobile Phases
times can be calculated relative to an internal standard
in pesticide residue analysis, and the results compared In normal-phase HPLC, the stationary phase is a
to a stored database of standards that the software polar adsorbant, such as bare silica or silica to which
automatically accesses when the analysis is complete. polar nonionic functional groups – hydroxyl, nitro,
The software will then assign and integrate the peaks, cyano (nitrile), or amino – have been chemically
and construct a complete report that will be displayed attached. These bonded phases are moderately polar
when the file is opened. and the surface is more uniform, resulting in better
The data stations are now more than simply peak shapes. The mobile phase for this mode con-
for interpreting results: the software packages also sists of a nonpolar solvent, such as hexane, to which is
include all the parameters needed to run the HPLC, added a more polar modifier, such as methylene chlo-
including start and stop, injection of the sample, and ride, to control solvent strength and selectivity. Solvent

28-1
table Example Applications of HPLC in the Analysis of Various Food Constituents

Analyte Separation Mode Method of Detection Chapter Section

Mono- and Ion exchange; normal- or Electrochemical; refractive 10 10.3.4.1

oligosaccharides reversed phase index; postcolumn analysis
Vitamin E Normal or reversed phase Fluorescence; 11 11.2.5.2
electrochemical; UV
Amino acids Ion exchange; Post or precolumn 15 15.3.1
reversed-phase derivatization
Pesticides Normal or reversed phase UV; fluorescence; mass 18 18.3.3.2.3; 18.3.3.3.2
spectrometry
Mycotoxins Reversed phase; UV; fluorescence 18 18.4.3.2.1
immunoaffinity
Antibiotics Reversed phase UV 18 18.5.2.2
Melamine Reversed phase, ion UV; mass spectrometry 18 18.8.3
exchange
Sulfites Ion exchange UV; electrochemical 18 18.8.2
508 Part V • Chromatography

strength refers to the way a solvent affects the migra- 28.3.2.2 Applications of Reversed-Phase
tion rate of the sample. Weak solvents increase reten- HPLC
tion (large k′ values) and strong solvents decrease
Reversed-phase has been the HPLC mode most used
retention (small k′ values).
for analysis of plant proteins. Cereal proteins, among
the most difficult of these proteins to isolate and char-
28.3.1.2 Applications of Normal-Phase HPLC acterize, are now routinely analyzed by this method
In the past, normal-phase HPLC was used for the (10). Both water- and fat-soluble vitamins (Chap. 11)
analysis of fat-soluble vitamins, although reverse can be analyzed by reversed-phase HPLC (2–5), and
phase is currently applied more frequently for these the availability of fluorescence detectors has enabled
analyses (see Table 28-1). Normal phase is currently researchers to quantitate very small amounts of the
used for the analyses of biologically active polyphe- different forms of vitamin B6 (vitamers) in foods and
nols from natural plant sources, such as grape and
cocoa. It is also used for the analysis of relatively
polar vitamins, such as vitamins A, D, E, and K
(see Chap. 11), and also natural carotenoid pigments,
which impart both color and health benefits to foods.
Highly hydrophilic species, such as carbohydrates (see
Chap. 10, Sect. 10.3.4.1), also may be resolved by
normal-phase chromatography, using amino bonded-
phase HPLC columns (10).

28.3.2 Reversed Phase

28.3.2.1 Stationary and Mobile Phases
More than 70% of all HPLC separations are carried
out in the reversed-phase mode, which utilizes a non-
polar stationary phase and a polar mobile phase.
Octadecylsilyl (ODS) bonded phases, with an octade-
cyl (C18 ) chain [−(CH2 )17 CH3 ], are the most popular
reversed-phase packing materials, although shorter
chain hydrocarbons [e.g., octyl (C8 ) or butyl (C4 )]
or phenyl groups are also used. Many silica-based,
reversed-phase columns are commercially available.
Differences in their chromatographic behavior result
from variation in the type of organic group bonded to
the silica matrix or the chain length of organic moiety.
Reversed-phase HPLC utilizes polar mobile
phases, usually water mixed with methanol, acetoni-
trile, or tetrahydrofuran. Solutes are retained due
to hydrophobic interactions with the nonpolar sta-
tionary phase and are eluted in order of increas-
ing hydrophobicity (decreasing polarity). Increas-
ing the polar (aqueous) component of the mobile
phase increases solute retention (larger k’ values) (see
Chap. 27, Sect. 27.5.2.2.4), whereas increasing the
organic solvent content of the mobile phase decreases Analysis of vitamin B6 compounds by
28-4 reversed-phase HPLC with fluorescence
retention (smaller k’ values). Various additives can figure
detection. Some of the standard compounds
serve additional functions. For example, although (a) are present in a sample of rice bran extract
ionic compounds often can be resolved without them, (b). Sample preparation and analytical pro-
ion-pair reagents may be used to facilitate chromatog- cedures are described in reference (17). PL,
raphy of ionic species on reversed-phase columns. pyridoxal; PLP, pyridoxal phosphate; PM,
pyridoxamine; PMP, pyridoxamine phos-
These reagents are ionic surfactants, such as octanesul- phate; PN, pyridoxine; PNG, pyridoxine
fonic acid, which can neutralize charged solutes and β-D-glucoside. [Reprinted in part with per-
make them more lipophilic. This type of chromatogra- mission from (17). Copyright 1991 American
phy is referred to as ion-pair reversed-phase. Chemical Society.]
Chapter 28 • High-Performance Liquid Chromatography 509

biological samples. Figure 28-4 shows the separation 28.3.3 Ion Exchange
of several of these vitamers in a rice bran extract
28.3.3.1 Stationary and Mobile Phases
achieved by reversed-phase ion-pair HPLC (17).
Reversed-phase ion-pair HPLC can be used to Packing materials for ion-exchange HPLC are usu-
resolve carbohydrates on C18 bonded-phase columns ally functionalized organic resins, such as sulfonated
(10), and the constituents of soft drinks (caffeine, or aminated poly(styrene-divinylbenzene) (Chap. 27,
aspartame, etc.) can be rapidly separated. Reversed- Sect. 27.4.3). Macroporous resins are most effective
phase HPLC using a variety of detection methods, for HPLC columns due to their rigidity and per-
including RI, UV, and light scattering, has been manent pore structure. Pellicular packings also are
applied to the analysis of lipids (2–5,10). Antioxidants, TM
utilized, particularly in the CarboPac (Dionex)
such as butylated hydroxyanisole (BHA) and buty- series, in which the nonporous, latex resin beads are
lated hydroxytoluene (BHT), can be extracted from dry coated with functionalized microbeads. The mobile
foods and analyzed with simultaneous UV and flu- phase in ion-exchange HPLC is usually an aqueous
orescence detection (3). Phenolic flavor compounds buffer, and solute retention is controlled by changing
(such as vanillin) and pigments (such as chlorophylls, mobile phase ionic strength and/or pH. Gradient elu-
carotenoids, and anthocyanins) are also easily ana- tion (gradually increasing ionic strength) is frequently
lyzed (2–5,10). A typical chromatogram of carotenoids employed.
present in a carrot extract is shown in Fig. 28-5.
Reversed-phase ion-pair chromatography also is used
for the separation of synthetic food colors (e.g., FD&C 28.3.3.2 Applications of Ion-Exchange HPLC
Red No. 40 and FD&C Blue No. 1) (5). Ion-exchange HPLC has many applications, ranging
from the detection of simple inorganic ions, to analysis
of carbohydrates and amino acids, to the preparative
purification of proteins oligosaccharides.

28.3.3.2.1 Ion Chromatography Ion chromatogra-

phy is simply high-performance ion-exchange chro-
matography using a relatively low-capacity stationary
phase (either anion- or cation-exchange) and, usually,
a conductivity detector. All ions conduct an electric
current; thus, measurement of electrical conductiv-
ity is an obvious way to detect ionic species. Because
the mobile phase also contains ions, however, back-
ground conductivity can be relatively high. One step
toward solving this problem is to use much lower
capacity ion-exchange packing materials, so that more
dilute eluents may be employed. In nonsuppressed or
single-column ion chromatography, the detector cell
is placed directly after the column outlet and eluents
are carefully chosen to maximize changes in conduc-
tivity as sample components elute from the column.
Suppressed ion chromatography utilizes an eluent
that can be selectively removed by the use of ion-
exchange membranes (10). Suppressed ion chromatog-
Reversed-phase HPLC separation of raphy permits the use of more concentrated mobile
28-5 α-carotene (AC) and β-carotene (BC) iso- phases and gradient elution. Ion chromatography can
figure
mers in (a) fresh and (b) canned carrots using be used to determine inorganic anions and cations,
a 5 µm C30 stationary phase. Peak 1, 13-cis transition metals, organic acids, amines, phenols, sur-
AC; 2, unidentified cis AC; 3, 13′ -cis AC; 4,
factants, and sugars. Some specific examples of ion
15-cis BC; 5, Unidentified cis AC; 6, 13-cis BC;
7, all-trans AC; 8, 9-cis AC; 9, all-trans BC; 10, chromatography applied to food matrices include the
9-cis BC. [Reprinted with permission from (18). determination of organic and inorganic ions in milk;
Copyright 1997 American Chemical Society.] organic acids in coffee extract and wine; chlorine in
510 Part V • Chromatography

Anion exchange analysis of iso-amylase-

Ion-chromatographic analysis of organic 28-7 treated waxy corn starch. The enzyme
28-6 acids and inorganic anions in coffee. Ten figure
debranches the amylopectin, and the chro-
figure
anions (listed) were resolved on an IonPac matogram represents the branch chain-length
AS5A column (Dionex) using a sodium distribution, from four sugars in length and
hydroxide gradient and suppressed conduc- up. The analysis was performed with anion-
tivity detection. (Courtesy of Dionex Corp., exchange HPLC (Dionex
TM
), with a pulsed
Sunnyvale, CA.) amperometric detector.

infant formula; and trace metals, phosphates, and 28.3.4 Size Exclusion
sulfites in foods. Figure 28-6 illustrates the simulta-
neous determination of organic acids and inorganic Size-exclusion chromatography (SEC) fractionates
anions in coffee by ion chromatography. solutes solely on the basis of size, with larger
molecules eluting first. Due to the limited separation
volume available in this chromatographic mode, as
explained in Chap. 27 (Sect. 27.4.4), the peak capac-
28.3.3.2.2 Ion Exchange Chromatography of Car- ity of a size-exclusion column is relatively small. Thus,
bohydrates and Proteins Both cation- and anion- the “high-performance” aspect of HPLC is not really
exchange stationary phases have been applied to applicable in the case of size exclusion. The main
HPLC of carbohydrates. The advantage of separating advantage gained from use of small particle pack-
carbohydrates by anion exchange is that retention and ing materials is speed. Relatively small amounts of
selectivity may be altered by changes in eluent com- sample can be analyzed or separated and collected in
position. Carbohydrate analysis has benefited greatly ≤60 min, compared to ≤24 h separations using clas-
by the development of a system that involves anion- sic low pressure systems (10). A second advantage is
exchange HPLC at high pH (≥12), and detection that the sample concentration is higher and the rela-
by a pulsed amperometric detector (PAD). Pellicular tive volume is lower, so there is much less eluent to
column packings (see Sect. 28.2.3.2.2), consisting of remove.
nonporous latex beads coated with a thin film of strong
anion exchanger, provide the necessary fast exchange,
high efficiency, and resistance to strong alkali. These 28.3.4.1 Column Packings and Mobile
systems may be used in a variety of applications, Phases
from routine quality control to basic research. One Size-exclusion packing materials or columns are
common application is the determination of oligosac- selected so that matrix pore size matches the molecular
charide distributions in corn syrups and other starch weight range of the species to be resolved. Prepacked
hydrolysates (Fig. 28-7). columns of microparticulate media are available in a
Amino acids have been resolved on polymeric wide variety of pore sizes. Hydrophilic packings, for
ion exchangers for more than 40 years (see Chap. 15, use with water-soluble samples and aqueous mobile
Sect. 15.3.1.2). Ion exchange is one of the most effec- phases, may be surface-modified silica or methacrylate
tive modes for HPLC of proteins and, recently, has resins. Poly(styrene-divinylbenzene) resins are use-
been recognized as valuable for the fractionation of ful for nonaqueous size-exclusion chromatography of
peptides. synthetic polymers.
Chapter 28 • High-Performance Liquid Chromatography 511

The mobile phase in this mode is chosen for has been shown to be a rapid, one-step method for
sample solubility, column compatibility, and minimal assessing soybean cultivars on the basis of protein con-
solute–stationary phase interaction. Otherwise, it has tent (proteins in the extracts of nondefatted flours from
little effect on the separation. Aqueous buffers are used five soybean cultivars were separated into six common
for biopolymers, such as proteins and polysaccharides, peaks, and cultivars could be identified by the per-
both to preserve biological activity and to prevent cent total area of the fifth peak). A size-exclusion liquid
adsorptive interactions. Tetrahydrofuran or dimethyl- chromatographic method also has been applied to the
formamide is generally used for size-exclusion chro- determination of polymerized triacylglycerols in oils
matography of other polymer samples, to ensure and fats (2).
sample solubility.

28.3.5 Affinity
28.3.4.2 Applications of High
Performance SEC Affinity chromatography is based on the principle
that the molecules to be purified can form a selec-
Hydrophilic polymeric size-exclusion packings are tive but reversible interaction with another molecular
used for the rapid determination of average molecu- species that has been immobilized on a chromato-
lar weight and molecular weight range of polysac- graphic support. Although almost any material can
charides, including amylose, amylopectin, and other be immobilized on a suitably activated support, the
soluble gums such as xanthan, pullulan, guar, and major ligands are proteins, including lectins (Chap. 15,
water-soluble cellulose derivatives. Molecular weight Sect. 15.2.3.2.2), nucleic acids, and dyes (Chap. 27,
distribution can be determined directly from high per- Sect. 27.4.5). Affinity chromatography is used to purify
formance size-exclusion chromatography, if LALLS many glycoproteins. Affinity chromatography using
or MALLS is used for detection (8, 10). The appli- immobilized folate-binding protein is an effective tool
cation of aqueous size-exclusion chromatography to in purifying sample extracts for HPLC analysis of
two commercially important polysaccharides, xanthan folates in foods [e.g., reference (19)].
and carboxymethyl cellulose, is discussed in detail in
reference (8).
SEC analysis has been useful to better understand
numerous food components and systems. SEC analy- 28.4 SUMMARY
sis of tomato cell-wall pectin from hot-and cold-break
tomato preparations (Fig. 28-8) showed that the cell- HPLC is a chromatographic technique of great ver-
wall pectin was not differentially degraded by the satility and analytical power. A basic HPLC system
different processing procedures. Size-exclusion HPLC consists of a pump, injector, column, detector, and data
system. The pump delivers mobile phase through the
system. An injector allows sample to be placed into
the flowing mobile phase for introduction onto the
column. The HPLC column consists of stainless steel
or polymer hardware filled with a separation pack-
ing material. Various auxiliary columns, particularly
guard columns, may be used prior to the analytical col-
umn. Detectors used in HPLC include UV-Vis absorp-
tion, fluorescence, RI, electrochemical, and light scat-
tering, as well as coupled analytical systems, such as a
mass spectrometer. Detection sensitivity or specificity
sometimes can be enhanced by chemical derivatiza-
tion of the analyte. Computer-controlled data station
systems offer data collection and processing capabili-
ties, and can run the instrument when an automated
system is needed. A broad variety of column packing
Analysis of tomato cell-wall pectin from materials have contributed greatly to the widespread
28-8 hot- and cold-break tomato preparations by use of HPLC. These column packing materials may
figure
size-exclusion chromatography. The solid lines be categorized as silica-based (porous silica, bonded
are from a refractive index detector response.
The lines with markers result from a postchro-
phases, pellicular packings) or polymeric (microp-
matography analysis. Aliquots of collected orous, macroporous, or pellicular/nonporous). The
fractions were analyzed by a colorimetric success of silica-based bonded phases has expanded
chemical assay that is specific for pectic sugars. the applications of normal-phase and reversed-phase
512 Part V • Chromatography

modes of separation in HPLC. Separations also are 28.7 REFERENCES

achieved with ion-exchange, size-exclusion, and affin-
ity chromatography. HPLC is widely used for the 1. Bidlingmeyer BA (1993) Practical HPLC methodology
analysis of small molecules and ions, such as sugars, and applications. Wiley, New York
vitamins, and amino acids, and is applied to the sep- 2. Chang SK, Holm E, Schwarz,J, Rayas-Duarte P (1995)
aration and purification of macromolecules, such as Food (applications review). Anal Chem 67:127R–153R
proteins and polysaccharides. 3. Matissek R, Wittkowski R (eds) (1993) High performance
liquid chromatography in food control and research.
Technomic Publishing, Lancaster, PA
4. Nollet LML (ed) (2000) Food analysis by HPLC, 2nd edn.
28.5 STUDY QUESTIONS Marcel Dekker, New York
5. Macrae R (ed) (1988) HPLC in food analysis, 2nd edn.
1. Why might you choose to use HPLC rather than tradi- Academic, New York, NY
tional low-pressure column chromatography? 6. Harris DC (2006) Quantitative chemical analysis, 7th
2. What is a guard column and why is it used? edn. W.H. Freeman and Co., New York
3. Give three general requirements for HPLC column pack- 7. Hanai T (2004) HPLC: a practical guide. The Royal
ing materials. Describe and distinguish among porous Society of Chemistry, Cambridge
silica, bonded phases, pellicular, and polymeric column 8. Swadesh J (ed) (2000) HPLC: practical and industrial
packings, including the advantages and disadvantages of applications, 2nd edn. CRC, Boca Raton, FL
each type. 9. Lough WJ, Wainer IW (eds) (2008) High performance liq-
4. What is the primary function of an HPLC detector uid chromatography: fundamental principles and prac-
(regardless of type)? What factors would you consider tice. Springer, New York
in choosing an HPLC detector? Describe three different 10. Heftmann E (ed) (1992) Chromatography, 5th edn. Fun-
types of detectors and explain the principles of operation damentals and applications of chromatography and
for each. related differential migration methods. Part A: funda-
5. You are performing HPLC using a stationary phase that mentals and techniques. Part B: applications. J Chro-
contains a polar nonionic functional group. What type matogr Libr Ser Vols. 51A and 51B. Elsevier, Amsterdam
of chromatography is this, and what could you do to 11. Gertz C (1990) HPLC tips and tricks. LDC Analytical,
increase the retention time of an analyte? Riviera Beach, FL
6. Why are external standards commonly used for HPLC 12. Dolan JW, Snyder LR (1989) Troubleshooting HPLC
(unlike in GC, for which internal standards are more systems: a systematic approach to troubleshooting LC
commonly used)? equipment and separations. Humana, Clifton, NJ
7. Ion chromatography has recently become a widely 13. Ishii D (ed) (1988) Introduction to microscale high-
promoted chromatographic technique in food analysis. performance liquid chromatography. VCH Publishers,
Describe ion chromatography and give at least two exam- New York
ples of its use. 14. Dorsey JG, Cooper WT, Siles BA, Foley JP, Barth HG
8. Describe one application each for ion-exchange and size- (1996) Liquid chromatography: theory and methodology
exclusion HPLC. (fundamental review). Anal Chem 68:515R–568R
15. LaCourse WR (2000) Column liquid chromatography:
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Anal Chem 72:37R–51R
16. Unger KK (1990) Packings and stationary phases in
28.6 ACKNOWLEDGMENTS chromatographic techniques. Marcel Dekker, New York
17. Gregory JF, Sartain DB (1991) Improved chromato-
Mary Ann Rounds (deceased) has been included as an graphic determination of free and glycosylated forms of
author in this chapter, in recognition of the key role vitamin B6 in foods. J Agric Food Chem 39:899–905
she played in writing the chapter on HPLC for the 1st– 18. Lessin WJ, Catignani GL, Schwartz SJ (1997) Quantifica-
3rd editions of this textbook. Much of her writing was tion of cis-transisomers of provitamin A carotenoids in
included in this chapter for the 4th edition. Dr. Reuhs, fresh and processed fruits and vegetables. J Agric Food
Chem 45:3728–3732
the primary author of this chapter, also wishes to
19. Pfeiffer C, Rogers LM, Gregory JF (1997) Determination
acknowledge the involvement of Dr. Jesse F. Gregory,
of folate in cereal-grain food products using tri-enzyme
III, with this chapter for previous editions of the book. extraction and combined affinity and reverse-phase liq-
Dr. Baraem Ismail is acknowledged for her thought- uid chromatography. J Agric Food Chem 45:407–413
ful suggestions on reorganizing the chromatography 20. Synder LR, Glajch JL, Kirkland JJ (1997) Practical HPLC
chapters, and her preparation of Table 28-1. method development, 2nd edn. Wiley, New York