INTRODUCTION

High-performance liquid chromatography (or High pressure liquid

chromatography, HPLC) is a form of column chromatography used frequently
inbiochemistry and analytical chemistry to separate, identify, and quantify compounds.
HPLC utilizes a column that holds chromatographic packing material (stationary phase),
a pump that moves the mobile phase(s) through the column, and a detector that shows
the retention times of the molecules. Retention time varies depending on the
interactions between the stationary phase, the molecules being analyzed, and the
solvent(s) used.

OPERATION

The sample to be analyzed is introduced in small volume to the stream of mobile

phase. The analyte's motion through the column is slowed by specific chemical or
physical interactions with the stationary phase as it traverses the length of the column.
The amount of retardation depends on the nature of the analyte, stationary phase and
mobile phase composition. The time at which a specific analyte elutes (comes out of
the end of the column) is called the retention time; the retention time under particular
conditions is considered a reasonably unique identifying characteristic of a given
analyte. The use of smaller particle size column packing (which creates higher
backpressure) increases the linear velocitygiving the components less time
to diffuse within the column, leading to improved resolution in the
resultingchromatogram. Common solvents used include any miscible combination
of water or various organic liquids (the most common are methanol and acetonitrile).
Water may contain buffers or salts to assist in the separation of the analyte
components, or compounds such as trifluoroacetic acid which acts as an ion pairing
agent.

1
MODES OF SEPARATION

Normal-Phase Chromatography

Normal-phase chromatography, or NP, is the classic form of liquid chromatography

using polar stationary phases and non-polar mobile phases. The analyte is retained by
the interaction of its polar functional groups with the polar groups on the surface of the
packing. Analytes elute from the column starting with the least polar compound
followed by other compounds in order of their increasing polarity. Normal-phase
chromatography is useful in the separation of analytes with low to intermediate polarity
and high solubility in low-polarity solvents. Water-soluble analytes are usually not good
candidates for normal-phase chromatography.

Reversed-Phase Chromatography

2
Reversed-phase chromatography, or RP, has become the most common mode of liquid
chromatographic separation. In RP the stationary phase is non-polar and the mobile
phase is polar. The analytes are attracted to the surface by their non-polar functional
groups. The most polar analyte elutes from the RP column first followed by other
analytes in order of decreasing polarity. RP chromatography is useful for the separation
of compounds having high to intermediate polarity.

Ion-Exchange Chromatography

Ion-exchange chromatography separates analytes by their ionic functionality. Ion-

exchange stationary phases are usually comprised of ionic species attached to the
surface of the silica substrate or ionic functional groups evenly distributed throughout a
polymeric media. Weak ion-exchange phases are usually pH dependent. Strong ion-
exchange phases are always charged and therefore independent of typical pH changes.
Ion-exchange columns with low capacity are used for ion chromatographic applications
where low ionic strength mobile phases are required for conductivity detection.

Affinity Chromatography

Affinity chromatography is based on specific interactions in a lock-and-key paradigm

between analytes and matrix-bound ligands. Dependent on the secondary structures of
biological macromolecultes for retention of selected sample components, affinitiy
chromatography is without question the most highly specific, and consequently the
most powerful, mode of chromatography.

Isocratic flow and gradient elution

A separation in which the mobile phase composition remains constant throughout the
procedure is termed isocratic (meaning constant composition). The word was coined by
Csaba Horvath from Yale University[citation needed], 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.[2] 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.
Solvent A is often water, 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.

3
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.[citation needed]

The driving force is originated in reversed phase chromatography in the high order of
the water structure. The role of the organic mobile phase is to reduce this high order by
reducing the retarding strength of the aqueous component.

Substrate Materials

Silica

Porous silica particles are the most common substrate material used for HPLC column
packings. Silica-based columns can withstand high pressures, are compatible with most
organic and aqueous mobile-phase solvents, and come in a wide range of bonded
phases. Silica-based columns are often used for separations of low molecular weight
analytes using mobile phase solvents and samples with a pH range of 2 to 7.5. Some
new-generation silica materials, such as EVEREST™, DENALI™, and GENESIS™, have
extended pH ranges.

Polymeric

Highly cross-linked styrene-divinylbenzene based packings are compatible with most

mobile phase solvents and samples with a pH of 1 to 14. Polymer-based columns tend
to have lower efficiencies for small molecules compared to silica-based columns due to
their smaller average surface area. In addition, polymer-based columns typically have
lower mechanical strength and therefore cannot withstand the highest system
backpressures. A polymer-based packing is often a good alternative if the sample
requires a mobile phase pH outside the normal operating range of standard silic-based
columns. Polymeric packings are often used for ion-exchange separations, and are also
useful in non-aqueous GPC size-exclusing analyses and ion exclusion analyses of
organic acids and carbohydrates.

Particle Properties

4
Irregular Shape

The first available HPLC columns were packed using irregularly shaped silica particles.
Because of this, many standard analytical methods are still based on these materials.
Irregular particles are also used in large- scale preparative applications because of their
high surface area, capacity and low cost.

Spherical Shape

The majority of new HPLC methods are performed on spherical shaped or spheroidal
(almost spherical) particles. Spherical particles provide higher efficiency, better column
stability and lower back-pressures compared to irregularly shaped particles.

Particle Size

Particle size for HPLC column packings refers to the average diameter of the packing
particles. Most HPLC packings contain a narrow range of particle diameters. Particle
size affects the back-pressure of the column and the separation efficiency. Column
back-pressure and column efficiency are inversely proportional to the square of the
particle diameter. This means that as the particle size decreases, the column back-
pressure and efficiency increase. A well packed column with 3 μm packings produces
almost twice the separation efficiency of a comparable 5 μm column. However, the 3
μm column will have about a three-fold higher back-pressure compared to the 5 μm
column when operated with the same mobile phase and at the same flow rate. Highly
efficient, small-particle (3 μm and 4 μm) columns are ideal for complex mixtures with
similar components. Fast, high-resolution separations can be achieved with small
particles packed in short (10-50 mm length) columns. Grace Vydac offers SHORTFAST™
and LIGHTNING™ HPLC columns specifically for these fast-HPLC applications.

Larger particle (5 μm and 7 μm) columns are typically used for routine analyses where
analytes have greater structural differences. Large 10 μm packings have only moderate
column efficiencies. Columns packed with 10 μm packings are generally used as scout
columns for future preparative separations, semi- preparative applications, or routine
QA/QC methods where high chromatographic efficiencies are not required. Large
particles (15- 20 μm) are used for preparative-scale separations.

Pore Size

The pore size of a packing material represents the average size of the pores within
each particle. The size of the analyte should be considered when choosing the
appropriate pore size for the packing material. The molecular weight of an analyte can
be used to estimate the size of the molecule. As a general rule, a pore size of 100 Å or
less should be used for analytes below 3,000 MW. A pore size of 100 Å -130 Å is
recommended for samples in the range of 3,000 MW - 10,000 MW. For samples
above10,000MW, including peptides and proteins, a 300 Å material provides the best
efficiency and peak shape.

5
Pore Volume

Pore volume is a measurement of the empty space within a particle. Pore volume is a
good indicator of the mechanical strength of a packing. Particles with large pore
volumes are typically weaker than particles with small pore volumes. Pore volumes of
1.0 mL/g or less are recommended for most HPLC separations. Pore volumes of greater
than 1.0mL/g are preferred for size-exclusion chromatography and useful for low-
pressure methods.

Surface Area

The physical structure of the particle substrate determines the surface area of the
packing material. Surface area is determined by pore size. Pore size and surface area
are inversely related. A packing material with a small pore size will have a large surface
area, and vice versa. High surface area materials offer greater capacity and longer
analyte retention times. Low surface area packings offer faster equilibration time and
are often used for large molecular weight molecules.

Carbon Load

The carbon load is a measure of the amount of bonded phase bound to the surface of
the packing. High carbon loads provide greater column capacities and resolution. Low
carbon loads produce less retentive packing and faster analysis times.

Surface Coverage

Surface coverage is calculated from the carbon load and surface area of a packing
material. Surface coverage affects the retention, selectivity and stability of bonded
phases.

End-Capping

A reversed-phase HPLC column that is end-capped has gone through a secondary

bonding step to cover unreacted silanols on the silica surface. End-capped packing
materials eliminate unpredictable secondary interactions. Basic analytes tend to
produce asymmetric tailed peaks on non end-capped columns, requiring the addition of
modifiers to the mobile phase. Non end-capped materials exhibit different selectivity
than end-capped columns. This selectivity difference can enhance separations of polar
analytes by controlling the secondary silanol interactions.

6
SOLVENTS SYSTEM IN THE HPLC

7
INSTRUMENTATION
The design characteristics divided HPLC injectors into four types. Figure 1.
presents the injector flow diagram for each type.
Type 1 Injectors - use a completely filled sample loop to determine the injected
volume. These simple, reliable devices are six-port rotary valves. A syringe is used to
push or suck an excess of sample into a sample loop, filling it completely. Highly
precise injections are achieved because the loop volume determines the injected
volume.
Type 2 Injectors - use a microsyringe to transfer sample into the loop. The
sample size is always smaller than the loop volume, so it is the syringe which
determines the injected volume. No sample is trapped or wasted, but the precision is
not as high as type 1.
LOAD INJECTION LOAD INJECTION

8
Figure 1. Injector Flow Diagrams. I - Load; II - Injection. The view is from the stator
-rotor interface, where the flow switching takes place, as seen from the front of
the injector. The small circles represent the ports in the valve stator. The bold
arcs and radial lines represent the connecting passages in the rotor, which turn
600 clockwise when the injector is moved from the load position to inject
position. The large circles represent the needle port.

Type 3 Injectors - use both complete and partial filling methods, but trap some
sample. The loop is loaded by inserting the syringe into the needle port and dispensing
the contents. The syringe is left inserted in the port until after the valve is switched.
The switching action inserts the loop into the stream without exposing the syringe to
high pressure. In the injection position the syringe is removed and some sample remains
trapped in a connecting passage of the injector. There are three consequences of this
trapped volume: sample is wasted, the injector must be flushed after each injection
and the syringe reading is in error by the amount of trapped volume.
Type 4 Injectors - also uses both methods, but does not trap sample. This type
is similar to type 3 injector but it does not contain a connecting passage between
syringe needle tip and sample loop. It therefore not trap sample and there is no sample
waste, no syringe reading error and no need to flush between injections, except in
trace analysis.

9
10
11
APPLICATION OF HPLC

1. Xanthines are the important constituents of tea, it can be analyzed by HPLC using heptane/ ethanol
(100:10).
2. Theophyline is analyzed with HPLC using chloroform/ isopropanol/ acetic acid (84:15:1).
3. Heroine sample analyzed by HPLC using acetonitrile (75%)/ ammonium acetate (65:35).
4. Codeine: methanol/ ammonium acetate (70:30).
5. Withaferine and withanone : hexane/ isopropanaol (3:2).
6. Cardinoloides: digitonxins t-butanol/acetonitrile/heptane/water (204:93:712:10.4).
7. Diosgenin: acetic acid/ethanol/methylene chloride/hexane (0.2:7:3:30:62.8).
8. Stropanthus and lanatosides : methanol: water (3:7).

12