High
Performance Liquid Chromatography ( HPLC
) Primer
Brief
History and Definition
The origins
of Liquid Chromatography began in the
early 1900’s with the work of the
Russian botanist, Mikhail S. Tswett. His
famous studies focused on separating
compounds (leaf pigments), which were
extracted from plants using a solvent.
He filled
an open glass column with particles. Two
specific materials that he found useful
were powdered chalk (calcium carbonate)
and
alumina. He poured his sample
(solvent extract of homogenized plant
leaves) into the column. This was
followed by pure solvent. As the
“sample” passed down through the column
by gravity, different colored “bands”
could be seen separated, because some
moved faster than others.
He related these separated, different
colored “bands” to the different
“compounds” which were originally
contained in the sample. He had created
an analytical separation of these
compounds based on a chemical attraction
of some compounds to the particles
(Stationary Phase). The compounds that
were attracted to the particles slowed
down, while other compounds that were
attracted to the solvent (Mobile Phase)
moved faster. This process can be
described as follows: sample compounds
“distribute” or “partition” differently
between the moving “mobile
phase” and the “stationary
phase”, creating a separation of the
compounds.
Tswett
named his process “chromatography”
[from the Greek words “chroma”, meaning
“color”, and “graphy”, meaning “writing”
(literally “color writing”) to describe
his “colorful” experiment. Today, Liquid
Chromatography, in its various forms,
has become one of the most powerful
tools in Analytical Chemistry.
Types of
Liquid Chromatography (LC)
Liquid
chromatography can be performed in three
primary approaches. In all cases, the
“sample” must be dissolved into a liquid
that is then transported by the solvent
onto or into, the chromatographic
device.
Approach 1)
The sample is “spotted” onto, and then
flowed through a thin layer of
chromatographic particles fixed onto the
surface of glass plates. The sample
appears black, but is actually made up
of yellow, red, and blue dyes. The
bottom edge of the plate is placed in a
solvent. The flow is created by solvent
diffusing through the dry particle layer
(by capillary action) moving up the
glass plate. This is called “Thin Layer
Chromatography ("TLC”).
Approach 2)
The sample is “spotted” onto
paper to which solvent is added to
create flow. This is called “Paper
Chromatography”.
Notice the
difference in separation power for this
particular paper. The samples were
mixtures of
dyes, some of which were separated
and some of which were not separated. In
the main pattern, the "black" sample
containing yellow, red and blue dyes was
applied to the paper. We see a green
ring and red ring. The paper could
not separate the yellow and blue, which
appear as green, but it could separate
those dyes from the red dye. The bottom
example is a sample of yellow and blue
only. Notice that the paper does not
separate those two dyes. The middle
example is a sample of red and blue
dyes. They are well separated. The
chromatographer has many different
statioary phases to chose from in order
to obtain the desired separation.
Approach 3)
In the most powerful approach, the
sample passes through a
column or a device containing
appropriate particles. These particles
are called the chromatographic packing
material, stationary phase or
“adsorbent”. Solvent flows continuously
through the column. At a point in time,
an “injection” of the sample solution is
made into the solvent stream, which then
carries the sample through the column.
As in Tswett’s experiment, the compounds
in the sample can then be separated by
traveling at different individual speeds
through the device.
When the
column or cartridge format is utilized,
there are several ways to achieve flow.
Gravity or vacuum can be used for
columns that are not designed to handle
pressure. Typically, the particles are
larger in size (>50 microns) to allow
flow to be generated more easily, as
with open glass columns (Tswett’s
experiment). In addition, plastic
“columns”, typically in the shape of
syringe barrels, can be filled with
packing material particles and used to
perform sample preparation. This is
called “Solid
Phase Extraction” (SPE).
Here, the chromatographic device, called
a “Cartridge”, is used usually with
vacuum to clean up a very complex sample
before it is analyzed further. (more
info...SPE
PRIMER &
OASIS)
For
improved separation power, smaller
particle sizes (<10 microns) are
required. These cause greater resistance
to flow, resulting in higher pressures
needed to create the solvent flow
required. Columns and pumps which are
designed to withstand high pressure are
necessary. When moderate to high
pressure is used to flow the solvent
through the chromatographic column, this
is called “HPLC”.
What is
High Performance Liquid Chromatography (HPLC)?
The name
“HPLC” originally referred to the fact
that high pressure was needed to
generate the flow required for liquid
chromatography in packed columns. In the
beginning, instrument components only
had the capability of generating
pressures of 500psi (35 bar). This was
called High Pressure Liquid
Chromatography (HPLC). The early 1970’s
saw a tremendous leap in technology.
These new “HPLC” instruments could
develop up to 6,000psi (400 bar) of
pressure, and included improved
detectors and columns. HPLC really began
to take hold in the mid to late 1970’s.
With continued advances in performance,
the name was changed to High Performance
Liquid Chromatography (HPLC). High
Performance Liquid Chromatography (HPLC)
is now one of the most powerful tools in
analytical chemistry, with the ability
to separate, identify and quantitate the
compounds that are present in any sample
that can be dissolved in a liquid.
Today, trace concentrations of
compounds, as low as “parts per
trillion” (ppt), are easily obtained.
HPLC can be applied to just about any
sample, such as pharmaceuticals, food,
nutraceuticals, cosmetics, environmental
matrices, forensic samples, and
industrial chemicals.
What is
Ultra Performance Liquid Chromatography
(UPLC™Technology)?
Beginning
in 2004, further advances in
instrumentation and column technology
were made to achieve very significant
increases in Resolution, Speed and
Sensitivity in Liquid Chromatography.
Columns with smaller particle sizes (1.7
micron) and instrumentation designed to
deliver 15,000psi (1,000 bar) along with
specialized capabilities were needed to
achieve a new level of performance. This
new technology is called ULTRA
PERFORMANCE LIQUID CHROMATOGRAPHY (UPLC™Technology).
A glimpse at what may be the future has
basic research being carried out today
by scientists working with columns
containing 1 micron particles, and
instrumentation capable of performing at
100,000psi (6,800 bar). |
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