High-performance liquid chromatography

High-performance liquid chromatography (HPLC; formerly referred to

as high-pressure liquid chromatography) is a technique in analytical
chemistry used to separate, identify, and quantify each component in a
mixture. It relies on pumps to pass a pressurized liquid solvent containing
the sample mixture through a column filled with a solid adsorbent material.
Each component in the sample interacts slightly differently with the
adsorbent material, causing different flow rates for the different
components and leading to the separation of the components as they flow
out of the column.
HPLC has been used for manufacturing (e.g., during the production
process of pharmaceutical and biological products), legal (e.g., detecting
performance enhancement drugs in urine), research (e.g., separating the
components of a complex biological sample, or of similar synthetic
chemicals from each other), and medical (e.g., detecting vitamin D levels in
blood serum) purposes.
Chromatography can be described as a mass transfer process
involving adsorption. HPLC relies on pumps to pass a pressurized liquid
and a sample mixture through a column filled with adsorbent, leading to the
separation of the sample components. The active component of the
column, the adsorbent, is typically a granular material made of solid
particles (e.g., silica, polymers, etc.), 2–50 μm in size. The components of
the sample mixture are separated from each other due to their different
degrees of interaction with the adsorbent particles. The pressurized liquid is
typically a mixture of solvents (e.g., water, acetonitrile and/or methanol)
and is referred to as a "mobile phase". Its composition
and temperature play a major role in the separation process by influencing
the interactions taking place between sample components and adsorbent.
These interactions are physical in nature, such as hydrophobic (dispersive),
dipole–dipole and ionic, most often a combination.
HPLC is distinguished from traditional ("low pressure") liquid
chromatography because operational pressures are significantly higher
(50–350 bar), while ordinary liquid chromatography typically relies on the
force of gravity to pass the mobile phase through the column. Due to the
small sample amount separated in analytical HPLC, typical column
dimensions are 2.1–4.6 mm diameter, and 30–250 mm length. Also HPLC
columns are made with smaller adsorbent particles (2–50 μm in average
particle size). This gives HPLC superior resolving power (the ability to
distinguish between compounds) when separating mixtures, which makes it
a popular chromatographic technique.
The schematic of a HPLC instrument typically includes a degasser,
sampler, pumps, and a detector. The sampler brings the sample mixture
into the mobile phase stream which carries it into the column. The pumps
deliver the desired flow and composition of the mobile phase through the
column. The detector generates a signal proportional to the amount of
sample component emerging from the column, hence allowing
for quantitative analysis of the sample components. A
digital microprocessor and user software control the HPLC instrument and
provide data analysis. Some models of mechanical pumps in a HPLC
instrument can mix multiple solvents together in ratios changing in time,
generating a composition gradient in the mobile phase. Various detectors
are in common use, such as UV/Vis, photodiode array (PDA) or based
on mass spectrometry. Most HPLC instruments also have a column oven
that allows for adjusting the temperature at which the separation is
performed.

High-performance liquid chromatography

An HPLC. From left to right: A pumping device

generating a gradient of two different solvents-
a steel-enforced column and a detector for
measuring the absorbance.
 A modern self-contained HPLC.

Schematic representation of an HPLC unit. (1) Solvent reservoirs, (2) Solvent degasser,
(3) Gradient valve, (4) Mixing vessel for delivery of the mobile phase, (5) High-pressure
pump, (6) Switching valve in "inject position", (6') Switching valve in "load position",
(7)Sample injection loop, (8) Pre-column (guard column), (9) Analytical column, (10)
Detector (i.e., IR, UV), (11) Data acquisition, (12) Waste or fraction collector.
 Contents
 1.Operation
 2.History and development
 3.Types
o 3.1Partition chromatography
o 3.2Normal–phase chromatography
o 3.3Displacement chromatography
o 3.4Reversed-phase chromatography (RPC)
o 3.5Size-exclusion chromatography
o 3.6Ion-exchange chromatography
o 3.7Bioaffinity chromatography
o 3.8Aqueous normal-phase chromatography
 4.Isocratic and gradient elution
 5.Parameters
o 5.1Theoretical
o 5.2Internal diameter
o 5.3Particle size
o 5.4Pore size
o 5.5Pump pressure
o 5.6Detectors
o 5.7Autosamplers
 6.Applications
o 6.1Manufacturing
o 6.2Legal
o 6.3Research
o 6.4Medical
Operation:
The sample mixture to be separated and analyzed is introduced, in a
discrete small volume (typically microliters), into the stream of mobile
phase percolating through the column. The components of the sample
move through the column at different velocities, which are a function of
specific physical interactions with the adsorbent (also called stationary
phase). The velocity of each component depends on its chemical nature,
on the nature of the stationary phase (column) and on the composition of
the mobile phase. The time at which a specific analyte elutes (emerges
from the column) is called its retention time. The retention time measured
under particular conditions is an identifying characteristic of a given
analyte.
Many different types of columns are available, filled with adsorbents
varying in particle size, and in the nature of their surface ("surface
chemistry"). The use of smaller particle size packing materials requires the
use of higher operational pressure ("backpressure") and typically improves
chromatographic resolution (the degree of peak separation between
consecutive analytes emerging from the column). Sorbent particles may be
hydrophobic or polar in nature.
Common mobile phases used include any miscible combination
of water with various organic solvents (the most common
are acetonitrile and methanol). Some HPLC techniques use water-free
mobile phases (see normal-phase chromatography below). The aqueous
component of the mobile phase may contain acids (such as formic,
phosphoric or trifluoroacetic acid) or salts to assist in the separation of the
sample components. The composition of the mobile phase may be kept
constant ("isocratic elution mode") or varied ("gradient elution mode")
during the chromatographic analysis. Isocratic elution is typically effective
in the separation of sample components that are very different in their
affinity for the stationary phase. In gradient elution the composition of the
mobile phase is varied typically from low to high eluting strength. The
eluting strength of the mobile phase is reflected by analyte retention times
with high eluting strength producing fast elution (=short retention times). A
typical gradient profile in reversed phase chromatography might start at 5%
acetonitrile (in water or aqueous buffer) and progress linearly to 95%
acetonitrile over 5–25 minutes. Periods of constant mobile phase
composition may be part of any gradient profile. For example, the mobile
phase composition may be kept constant at 5% acetonitrile for 1–3 min,
followed by a linear change up to 95% acetonitrile.

A rotary fraction collector collecting HPLC output. The system is being used
to isolate a fraction containing Complex I from E. coli plasma membranes.
About 50 litres of bacteria were needed to isolate this amount.
The chosen composition of the mobile phase (also called eluent) depends
on the intensity of interactions between various sample components
("analytes") and stationary phase (e.g., hydrophobic interactions in
reversed-phase HPLC). Depending on their affinity for the stationary and
mobile phases analytes partition between the two during the separation
process taking place in the column. This partitioning process is similar to
that which occurs during a liquid–liquid extraction but is continuous, not
step-wise. In this example, using a water/acetonitrile gradient, more
hydrophobic components will elute (come off the column) late, once the
mobile phase gets more concentrated in acetonitrile (i.e., in a mobile phase
of higher eluting strength).
The choice of mobile phase components, additives (such as salts or acids)
and gradient conditions depends on the nature of the column and sample
components. Often a series of trial runs is performed with the sample in
order to find the HPLC method which gives adequate separation.

History and development:

Prior to HPLC scientists used standard liquid chromatographic techniques.
Liquid chromatographic systems were largely inefficient due to the flow rate
of solvents being dependent on gravity. Separations took many hours, and
sometimes days to complete. Gas chromatography (GC) at the time was
more powerful than liquid chromatography (LC), however, it was believed
that gas phase separation and analysis of very polar high molecular
weight biopolymers was impossible. GC was ineffective for many
biochemists because of the thermal instability of the solutes. As a result,
alternative methods were hypothesized which would soon result in the
development of HPLC.
Following on the seminal work of Martin and Synge in 1941, it was
predicted by Cal Giddings, Josef Huber, and others in the 1960s that LC
could be operated in the high-efficiency mode by reducing the packing-
particle diameter substantially below the typical LC (and GC) level of 150
μm and using pressure to increase the mobile phase velocity. These
predictions underwent extensive experimentation and refinement
throughout the 60s into the 70s. Early developmental research began to
improve LC particles, and the invention of Zipax, a superficially porous
particle, was promising for HPLC technology.
The 1970s brought about many developments in hardware and
instrumentation. Researchers began using pumps and injectors to make a
rudimentary design of an HPLC system. Gas amplifier pumps were ideal
because they operated at constant pressure and did not require leak free
seals or check valves for steady flow and good quantitation. Hardware
milestones were made at Dupont IPD (Industrial Polymers Division) such
as a low-dwell-volume gradient device being utilized as well as replacing
the septum injector with a loop injection valve.
While instrumentational developments were important, the history of HPLC
is primarily about the history and evolution of particle technology. After the
introduction of porous layer particles, there has been a steady trend to
reduced particle size to improve efficiency. However, by decreasing particle
size, new problems arose. The practical disadvantages stem from the
excessive pressure drop needed to force mobile fluid through the column
and the difficulty of preparing a uniform packing of extremely fine materials.
Every time particle size is reduced significantly, another round of
instrument development usually must occur to handle the pressure.

Types:

Partition chromatography
HILIC Partition Technique Useful Range
Partition chromatography was one of the first kinds of chromatography that
chemists developed. The partition coefficient principle has been applied
in paper chromatography, thin layer chromatography, gas
phase and liquid–liquid separation applications. The 1952 Nobel Prize in
chemistry was earned by Archer John Porter Martin and Richard Laurence
Millington Synge for their development of the technique, which was used
for their separation of amino acids. Partition chromatography uses a
retained solvent, on the surface or within the grains or fibers of an "inert"
solid supporting matrix as with paper chromatography; or takes advantage
of some coulombic and/or hydrogen donor interaction with the stationary
phase. Analyte molecules partition between a liquid stationary phase and
the eluent. Just as in Hydrophilic Interaction Chromatography (HILIC; a
sub-technique within HPLC), this method separates analytes based on
differences in their polarity. HILIC most often uses a bonded
polar stationary phase and a mobile phase made primarily
of acetonitrilewith water as the strong component. Partition HPLC has been
used historically on unbonded silica or alumina supports. Each works
effectively for separating analytes by relative polar differences. HILIC
bonded phases have the advantage of separating acidic, basic and neutral
solutes in a single chromatographic run. The polar analytes diffuse into a
stationary water layer associated with the polar stationary phase and are
thus retained. The stronger the interactions between the polar analyte and
the polar stationary phase (relative to the mobile phase) the longer the
elution time. The interaction strength depends on the functional groups part
of the analyte molecular structure, with more polarized groups (e.g.,
hydroxyl-) and groups capable of hydrogen bonding inducing more
retention. Coulombic (electrostatic) interactions can also increase retention.
Use of more polar solvents in the mobile phase will decrease the retention
time of the analytes, whereas more hydrophobic solvents tend to increase
retention times.
Normal–phase chromatography
Normal–phase chromatography was one of the first kinds of HPLC that
chemists developed. Also known as normal-phase HPLC (NP-HPLC) this
method separates analytes based on their affinity for a polar stationary
surface such as silica, hence it is based on analyte ability to engage in
polar interactions (such as hydrogen-bonding or dipole-dipole type of
interactions) with the sorbent surface. NP-HPLC uses a non-polar, non-
aqueous mobile phase (e.g., Chloroform), and works effectively for
separating analytes readily soluble in non-polar solvents. The analyte
associates with and is retained by the polar stationary phase. Adsorption
strengths increase with increased analyte polarity. The interaction strength
depends not only on the functional groups present in the structure of the
analyte molecule, but also on steric factors. The effect of steric hindrance
on interaction strength allows this method to resolve (separate) structural
isomers.
The use of more polar solvents in the mobile phase will decrease the
retention time of analytes, whereas more hydrophobic solvents tend to
induce slower elution (increased retention times). Very polar solvents such
as traces of water in the mobile phase tend to adsorb to the solid surface of
the stationary phase forming a stationary bound (water) layer which is
considered to play an active role in retention. This behavior is somewhat
peculiar to normal phase chromatography because it is governed almost
exclusively by an adsorptive mechanism (i.e., analytes interact with a solid
surface rather than with the solvated layer of a ligand attached to the
sorbent surface; see also reversed-phase HPLC below). Adsorption
chromatography is still widely used for structural isomer separations in both
column and thin-layer chromatography formats on activated (dried) silica or
alumina supports.
Partition- and NP-HPLC fell out of favor in the 1970s with the development
of reversed-phase HPLC because of poor reproducibility of retention times
due to the presence of a water or protic organic solvent layer on the
surface of the silica or alumina chromatographic media. This layer changes
with any changes in the composition of the mobile phase (e.g., moisture
level) causing drifting retention times.
Recently, partition chromatography has become popular again with the
development of Hilic bonded phases which demonstrate improved
reproducibility, and due to a better understanding of the range of
usefulness of the technique.
Displacement chromatography
The basic principle of displacement chromatography is: A molecule with a
high affinity for the chromatography matrix (the displacer) will compete
effectively for binding sites, and thus displace all molecules with lesser
affinities. There are distinct differences between displacement and elution
chromatography. In elution mode, substances typically emerge from a
column in narrow, Gaussian peaks. Wide separation of peaks, preferably to
baseline, is desired in order to achieve maximum purification. The speed at
which any component of a mixture travels down the column in elution mode
depends on many factors. But for two substances to travel at different
speeds, and thereby be resolved, there must be substantial differences in
some interaction between the biomolecules and the chromatography
matrix. Operating parameters are adjusted to maximize the effect of this
difference. In many cases, baseline separation of the peaks can be
achieved only with gradient elution and low column loadings. Thus, two
drawbacks to elution mode chromatography, especially at the preparative
scale, are operational complexity, due to gradient solvent pumping, and low
throughput, due to low column loadings. Displacement chromatography has
advantages over elution chromatography in that components are resolved
into consecutive zones of pure substances rather than “peaks”. Because
the process takes advantage of the nonlinearity of the isotherms, a larger
column feed can be separated on a given column with the purified
components recovered at significantly higher concentration.
Reversed-phase chromatography (RPC)

A chromatogram of complex mixture (perfume water) obtained by reversed

phase HPLC
Reversed phase HPLC (RP-HPLC) has a non-polar stationary phase and
an aqueous, moderately polar mobile phase. One common stationary
phase is a silica which has been surface-modified with RMe2SiCl, where R
is a straight chain alkyl group such as C18H37 or C8H17. With such stationary
phases, retention time is longer for molecules which are less polar, while
polar molecules elute more readily (early in the analysis). An investigator
can increase retention times by adding more water to the mobile phase;
thereby making the affinity of the hydrophobic analyte for the hydrophobic
stationary phase stronger relative to the now more hydrophilic mobile
phase. Similarly, an investigator can decrease retention time by adding
more organic solvent to the eluent. RP-HPLC is so commonly used that it is
often incorrectly referred to as "HPLC" without further specification. The
pharmaceutical industry regularly employs RP-HPLC to qualify drugs
before their release.
RP-HPLC operates on the principle of hydrophobic interactions, which
originates from the high symmetry in the dipolar water structure and plays
the most important role in all processes in life science. RP-HPLC allows the
measurement of these interactive forces. The binding of the analyte to the
stationary phase is proportional to the contact surface area around the non-
polar segment of the analyte molecule upon association with the ligand on
the stationary phase. This solvophobic effect is dominated by the force of
water for "cavity-reduction" around the analyte and the C18-chain versus the
complex of both. The energy released in this process is proportional to
the surface tension of the eluent (water: 7.3×10−6 J/cm², methanol:
2.2×10−6 J/cm²) and to the hydrophobic surface of the analyte and the
ligand respectively. The retention can be decreased by adding a less polar
solvent (methanol, acetonitrile) into the mobile phase to reduce the surface
tension of water. Gradient elution uses this effect by automatically reducing
the polarity and the surface tension of the aqueous mobile phase during the
course of the analysis.
Structural properties of the analyte molecule play an important role in its
retention characteristics. In general, an analyte with a larger hydrophobic
surface area (C–H, C–C, and generally non-polar atomic bonds, such as S-
S and others) is retained longer because it is non-interacting with the water
structure. On the other hand, analytes with higher polar surface area
(conferred by the presence of polar groups, such as -OH, -NH2, COO− or -
NH3+ in their structure) are less retained as they are better integrated into
water. Such interactions are subject to steric effects in that very large
molecules may have only restricted access to the pores of the stationary
phase, where the interactions with surface ligands (alkyl chains) take place.
Such surface hindrance typically results in less retention.
Retention time increases with hydrophobic (non-polar) surface area.
Branched chain compounds elute more rapidly than their corresponding
linear isomers because the overall surface area is decreased. Similarly
organic compounds with single C–C bonds elute later than those with a
C=C or C–C triple bond, as the double or triple bond is shorter than a
single C–C bond.
Aside from mobile phase surface tension (organizational strength in eluent
structure), other mobile phase modifiers can affect analyte retention. For
example, the addition of inorganic salts causes a moderate linear increase
in the surface tension of aqueous solutions (ca. 1.5×10−7 J/cm² per Mol for
NaCl, 2.5×10−7 J/cm² per Mol for (NH4)2SO4), and because the entropy of
the analyte-solvent interface is controlled by surface tension, the addition of
salts tend to increase the retention time. This technique is used for mild
separation and recovery of proteins and protection of their biological activity
in protein analysis (hydrophobic interaction chromatography, HIC).
Another important factor is the mobile phase pH since it can change the
hydrophobic character of the analyte. For this reason most methods use
a buffering agent, such as sodium phosphate, to control the pH. Buffers
serve multiple purposes: control of pH, neutralize the charge on the silica
surface of the stationary phase and act as ion pairing agents to neutralize
analyte charge. Ammonium formate is commonly added in mass
spectrometry to improve detection of certain analytes by the formation of
analyte-ammonium adducts. A volatile organic acid such as acetic acid, or
most commonly formic acid, is often added to the mobile phase if mass
spectrometry is used to analyze the column effluent. Trifluoroacetic acid is
used infrequently in mass spectrometry applications due to its persistence
in the detector and solvent delivery system, but can be effective in
improving retention of analytes such as carboxylic acids in applications
utilizing other detectors, as it is a fairly strong organic acid. The effects of
acids and buffers vary by application but generally improve
chromatographic resolution.
Reversed phase columns are quite difficult to damage compared with
normal silica columns; however, many reversed phase columns consist of
alkyl derivatized silica particles and should never be used with
aqueous bases as these will destroy the underlying silica particle. They can
be used with aqueous acid, but the column should not be exposed to the
acid for too long, as it can corrode the metal parts of the HPLC equipment.
RP-HPLC columns should be flushed with clean solvent after use to
remove residual acids or buffers, and stored in an appropriate composition
of solvent. The metal content of HPLC columns must be kept low if the best
possible ability to separate substances is to be retained. A good test for the
metal content of a column is to inject a sample which is a mixture of 2,2'-
and 4,4'-bipyridine. Because the 2,2'-bipy can chelate the metal, the shape
of the peak for the 2,2'-bipy will be distorted (tailed) when metal ions are
present on the surface of the silica.

Size-exclusion chromatography
Size-exclusion chromatography (SEC), also known as gel permeation
chromatography or gel filtration chromatography, separates particles on the
basis of molecular size (actually by a particle's Stokes radius). It is
generally a low resolution chromatography and thus it is often reserved for
the final, "polishing" step of the purification. It is also useful for determining
the tertiary structure and quaternary structure of purified proteins. SEC is
used primarily for the analysis of large molecules such as proteins or
polymers. SEC works by trapping these smaller molecules in the pores of a
particle. The larger molecules simply pass by the pores as they are too
large to enter the pores. Larger molecules therefore flow through the
column quicker than smaller molecules, that is, the smaller the molecule,
the longer the retention time.
This technique is widely used for the molecular weight determination of
polysaccharides. SEC is the official technique (suggested by European
pharmacopeia) for the molecular weight comparison of different
commercially available low-molecular weight heparins.
Ion-exchange chromatography
In ion-exchange chromatography (IC), retention is based on the attraction
between solute ions and charged sites bound to the stationary phase.
Solute ions of the same charge as the charged sites on the column are
excluded from binding, while solute ions of the opposite charge of the
charged sites of the column are retained on the column. Solute ions that
are retained on the column can be eluted from the column by changing the
solvent conditions (e.g., increasing the ion effect of the solvent system by
increasing the salt concentration of the solution, increasing the column
temperature, changing the pH of the solvent, etc.).
Types of ion exchangers include polystyrene resins, cellulose
and dextran ion exchangers (gels), and controlled-pore glass or porous
silica. Polystyrene resins allow cross linkage which increases the stability of
the chain. Higher cross linkage reduces swerving, which increases the
equilibration time and ultimately improves selectivity. Cellulose and dextran
ion exchangers possess larger pore sizes and low charge densities making
them suitable for protein separation.
In general, ion exchangers favor the binding of ions of higher charge and
smaller radius.
An increase in counter ion (with respect to the functional groups in resins)
concentration reduces the retention time. A decrease in pH reduces the
retention time in cation exchange while an increase in pH reduces the
retention time in anion exchange. By lowering the pH of the solvent in a
cation exchange column, for instance, more hydrogen ions are available to
compete for positions on the anionic stationary phase, thereby eluting
weakly bound cations.
This form of chromatography is widely used in the following applications:
water purification, preconcentration of trace components, ligand-exchange
chromatography, ion-exchange chromatography of proteins, high-pH anion-
exchange chromatography of carbohydrates and oligosaccharides, and
others.
Bioaffinity chromatography
This chromatographic process relies on the property of biologically active
substances to form stable, specific, and reversible complexes. The
formation of these complexes involves the participation of common
molecular forces such as the Van der Waals interaction, electrostatic
interaction, dipole-dipole interaction, hydrophobic interaction, and the
hydrogen bond. An efficient, biospecific bond is formed by a simultaneous
and concerted action of several of these forces in the complementary
binding sites.
Aqueous normal-phase chromatography
Aqueous normal-phase chromatography (ANP) is a chromatographic
technique which encompasses the mobile phase region between reversed-
phase chromatography (RP) and organic normal phase chromatography
(ONP). This technique is used to achieve unique selectivity for hydrophilic
compounds, showing normal phase elution using reversed-phase solvents.
Isocratic and gradient elution:

At the ARS Natural Products Utilization Research Unit in Oxford, MS., a

support scientist (r) extracts plant pigments that will be analyzed by a plant
physiologist (l) using an HPLC system.
A separation in which the mobile phase composition remains constant
throughout the procedure is termed isocratic (meaning constant
composition). (The example of these the percentage of methanol
throughout the procedure will remain constant i.e 10%) The word was
coined by Csaba Horvath who was one of the pioneers of HPLC.
The mobile phase composition does not have to remain constant. A
separation in which the mobile phase composition is changed during the
separation process is described as a gradient elution. One example is a
gradient starting at 10% methanol and ending at 90% methanol after 20
minutes. The two components of the mobile phase are typically termed "A"
and "B"; A is the "weak" solvent which allows the solute to elute only
slowly, while B is the "strong" solvent which rapidly elutes the solutes from
the column. In reversed-phase chromatography, solvent A is often water or
an aqueous buffer, while B is an organic solvent miscible with water, such
as acetonitrile, methanol, THF, or isopropanol.
In isocratic elution, peak width increases with retention time linearly
according to the equation for N, the number of theoretical plates. This leads
to the disadvantage that late-eluting peaks get very flat and broad. Their
shape and width may keep them from being recognized as peaks.
A schematic of gradient elution. Increasing mobile phase strength
sequentially elutes analytes having varying interaction strength with the
stationary phase.
Gradient elution decreases the retention of the later-eluting components so
that they elute faster, giving narrower (and taller) peaks for most
components. This also improves the peak shape for tailed peaks, as the
increasing concentration of the organic eluent pushes the tailing part of a
peak forward. This also increases the peak height (the peak looks
"sharper"), which is important in trace analysis. The gradient program may
include sudden "step" increases in the percentage of the organic
component, or different slopes at different times – all according to the
desire for optimum separation in minimum time.
In isocratic elution, the selectivity does not change if the column
dimensions (length and inner diameter) change – that is, the peaks elute in
the same order. In gradient elution, the elution order may change as the
dimensions or flow rate change.
The driving force in reversed phase chromatography originates in the high
order of the water structure. The role of the organic component of the
mobile phase is to reduce this high order and thus reduce the retarding
strength of the aqueous component.

Parameters:
Theoretical
HPLC separations have theoretical parameters and equations to describe
the separation of components into signal peaks when detected by
instrumentation such as by a UV detector or a mass spectrometer. The
parameters are largely derived from two sets of chromatagraphic theory:
plate theory (as part of Partition chromatography), and the rate theory of
chromatography / Van Deemter equation. Of course, they can be put in
practice through analysis of HPLC chromatograms, although rate theory is
considered the more accurate theory.
They are analogous to the calculation of retention factor for a paper
chromatography separation, but describes how well HPLC separates a
mixture into two or more components that are detected as peaks (bands)
on a chromatogram. The HPLC parameters are the: efficiency factor(N),
the retention factor (kappa prime), and the separation factor (alpha).
Together the factors are variables in a resolution equation, which describes
how well two components' peaks separated or overlapped each other.
These parameters are mostly only used for describing HPLC reversed
phase and HPLC normal phase separations, since those separations tend
to be more subtle than other HPLC modes (e.g., ion exchange and size
exclusion).
Void volume is the amount of space in a column that is occupied by
solvent. It is the space within the column that is outside of the column's
internal packing material. Void volume is measured on a chromatogram as
the first component peak detected, which is usually the solvent that was
present in the sample mixture; ideally the sample solvent flows through the
column without interacting with the column, but is still detectable as distinct
from the HPLC solvent. The void volume is used as a correction factor.
Efficiency factor (N) practically measures how sharp component peaks on
the chromatogram are, as ratio of the component peak's area ("retention
time") relative to the width of the peaks at their widest point (at the
baseline). Peaks that are tall, sharp, and relatively narrow indicate that
separation method efficiently removed a component from a mixture; high
efficiency. Efficiency is very dependent upon the HPLC column and the
HPLC method used. Efficiency factor is synonymous with plate number,
and the 'number of theoretical plates'.
Retention factor (kappa prime) measures how long a component of the
mixture stuck to the column, measured by the area under the curve of its
peak in a chromatogram (since HPLC chromatograms are a function of
time). Each chromatogram peak will have its own retention factor
(e.g., kappa1 for the retention factor of the first peak). This factor may be
corrected for by the void volume of the column.
Separation factor (alpha) is a relative comparison on how well two
neighboring components of the mixture were separated (i.e., two
neighboring bands on a chromatogram). This factor is defined in terms of a
ratio of the retention factors of a pair of neighboring chromatogram peaks,
and may also be corrected for by the void volume of the column. The
greater the separation factor value is over 1.0, the better the separation,
until about 2.0 beyond which an HPLC method is probably not needed for
separation. Resolution equations relate the three factors such that high
efficiency and separation factors improve the resolution of component
peaks in a HPLC separation.
Internal diameter

Tubing on a nano-liquid chromatography (nano-LC) system, used for very

low flow capacities.
The internal diameter (ID) of an HPLC column is an important parameter
that influences the detection sensitivity and separation selectivity in
gradient elution. It also determines the quantity of analyte that can be
loaded onto the column. Larger columns are usually seen in industrial
applications, such as the purification of a drug product for later use. Low-ID
columns have improved sensitivity and lower solvent consumption at the
expense of loading capacity.
Larger ID columns (over 10 mm) are used to purify usable amounts of
material because of their large loading capacity.
Analytical scale columns (4.6 mm) have been the most common type of
columns, though smaller columns are rapidly gaining in popularity. They
are used in traditional quantitative analysis of samples and often use a UV-
Vis absorbance detector.
Narrow-bore columns (1–2 mm) are used for applications when more
sensitivity is desired either with special UV-vis
detectors, fluorescence detection or with other detection methods like liquid
chromatography-mass spectrometry
Capillary columns (under 0.3 mm) are used almost exclusively with
alternative detection means such as mass spectrometry. They are usually
made from fused silicacapillaries, rather than the stainless steel tubing that
larger columns employ.
Particle size
Most traditional HPLC is performed with the stationary phase attached to
the outside of small spherical silica particles (very small beads). These
particles come in a variety of sizes with 5 µm beads being the most
common. Smaller particles generally provide more surface area and better
separations, but the pressure required for optimum linear velocity increases
by the inverse of the particle diameter squared.
This means that changing to particles that are half as big, keeping the size
of the column the same, will double the performance, but increase the
required pressure by a factor of four. Larger particles are used in
preparative HPLC (column diameters 5 cm up to >30 cm) and for non-
HPLC applications such as solid-phase extraction.
Pore size
Many stationary phases are porous to provide greater surface area. Small
pores provide greater surface area while larger pore size has better
kinetics, especially for larger analytes. For example, a protein which is only
slightly smaller than a pore might enter the pore but does not easily leave
once inside.
Pump pressure
Pumps vary in pressure capacity, but their performance is measured on
their ability to yield a consistent and reproducible volumetric flow
rate. Pressure may reach as high as 60 MPa (6000 lbf/in2), or about
600 atmospheres. Modern HPLC systems have been improved to work at
much higher pressures, and therefore are able to use much smaller particle
sizes in the columns (<2 μm). These ultra high performance liquid
chromatography" systems or UHPLCs can work at up to 120 MPa
(17,405 lbf/in2), or about 1200 atmospheres. The term "UPLC" is a
trademark of the Waters Corporation, but is sometimes used to refer to the
more general technique of UHPLC.
Detectors
HPLC detectors fall into two main categories: universal or selective.
Universal detectors typically measure a bulk property (e.g., refractive index)
by measuring a difference of a physical property between the mobile phase
and mobile phase with solute while selective detectors measure a solute
property (e.g., UV-Vis absorbance) by simply responding to the physical
or chemical property of the solute. HPLC most commonly uses a UV-Vis
absorbance detector, however, a wide range of other chromatography
detectors can be used. A universal detector that complements UV-Vis
absorbance detection is the Charged aerosol detector (CAD). A kind of
commonly utilized detector includes refractive index detectors, which
provide readings by measuring the changes in the refractive index of the
effluent as it moves through the flow cell. In certain cases, it is possible to
use multiple detectors, for example LCMS normally combines UV-Vis with
a mass spectrometer.
Autosamplers
Large numbers of samples can be automatically injected onto an HPLC
system, by the use of HPLC autosamplers. In addition, HPLC autosamplers
have an injection volume and technique which is exactly the same for each
injection, consequently they provide a high degree of injection volume
precision.

Applications:
Manufacturing
HPLC has many applications in both laboratory and clinical science. It is a
common technique used in pharmaceutical development, as it is a
dependable way to obtain and ensure product purity. While HPLC can
produce extremely high quality (pure) products, it is not always the primary
method used in the production of bulk drug materials. According to the
European pharmacopoeia, HPLC is used in only 15.5% of
syntheses. However, it plays a role in 44% of syntheses in the United
States pharmacopoeia. This could possibly be due to differences in
monetary and time constraints, as HPLC on a large scale can be an
expensive technique. An increase in specificity, precision, and accuracy
that occurs with HPLC unfortunately corresponds to an increase in cost.
Legal
This technique is also used for detection of illicit drugs in urine. The most
common method of drug detection is an immunoassay.This method is
much more convenient. However, convenience comes at the cost of
specificity and coverage of a wide range of drugs. As HPLC is a method of
determining (and possibly increasing) purity, using HPLC alone in
evaluating concentrations of drugs is somewhat insufficient. With this,
HPLC in this context is often performed in conjunction with mass
spectrometry. Using liquid chromatography instead of gas chromatography
in conjunction with MS circumvents the necessity for derivitizing with
acetylating or alkylation agents, which can be a burdensome extra
step. This technique has been used to detect a variety of agents like doping
agents, drug metabolites, glucuronide conjugates, amphetamines, opioids,
cocaine, BZDs, ketamine, LSD, cannabis, and pesticides. Performing
HPLC in conjunction with Mass spectrometry reduces the absolute need for
standardizing HPLC experimental runs.
Research
Similar assays can be performed for research purposes, detecting
concentrations of potential clinical candidates like anti-fungal and asthma
drugs. This technique is obviously useful in observing multiple species in
collected samples, as well, but requires the use of standard solutions when
information about species identity is sought out. It is used as a method to
confirm results of synthesis reactions, as purity is essential in this type of
research. However, mass spectrometry is still the more reliable way to
identify species.
Medical
Medical use of HPLC can include drug analysis, but falls more closely
under the category of nutrient analysis. While urine is the most common
medium for analyzing drug concentrations, blood serum is the sample
collected for most medical analyses with HPLC. Other methods of detection
of molecules that are useful for clinical studies have been tested against
HPLC, namely immunoassays. In one example of this, competitive protein
binding assays (CPBA) and HPLC were compared for sensitivity in
detection of vitamin D. Useful for diagnosing vitamin D deficiencies in
children, it was found that sensitivity and specificity of this CPBA reached
only 40% and 60%, respectively, of the capacity of HPLC.While an
expensive tool, the accuracy of HPLC is nearly unparalleled.