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HPLC
GC
vs.
1 Solvent
Common mobile phases
include any miscible
combination of water with
various organic solvents
the most common being
acetonitrile and methanol.
Additives such as salts
and acids may also
be included for some
techniques.
1 gas supply
Typical carrier gases include
helium, nitrogen, argon,
hydrogen, and air and are
usually determined by
the detector being used.
Higher flow rates yield faster
analysis, but less separation
between analytes.
He
Pump
Ar
2 sample injector
Analysis starts with the injection of sample.
Split Injection Sample is introduced to
the heated space where fast evaporation
occurs; sample mixes with carrier gas and
a small portion is introduced onto the
column. Due to large loss of sample, split
injection is not suitable for trace analysis
and, depending on injector temperature,
thermal degradation can take place.
Splitless Injection Suitable for trace
analysis as the complete sample is
introduced although it's more complicated
as the oven temperature, solvent, and the
splitless time have to be carefully selected.
AUTOSAMPLER
Autosamplers ensure reliable, precise, and
accurate injection and support a wide range of
formats and sample throughputs.
Over 95% of HPLC systems from major
manufacturers ship with autosamplers, a
testament to their reliability and reproducibility.
4 HPLC COLUMN
The most common HPLC columns are
made from stainless steel, but they can
also be made out of glass, polymers,
or a combination of materials. Typical
HPLC columns are between 3 and 25 cm
long and have a diameter of 1 to 5 mm.
Particles that pack the columns have
a typical diameter between 3 to 5 nm.
Liquid chromatographic columns will
increase in efficiency when the diameter
of the packed particles inside the
column decreases.
3 Capillary column
5 Detector
oven
UV-Vis The
most commonly
used detector, its
response is specific
to a particular
compound
depending on
the presence of
light absorbing
functional
groups of eluting
molecules.
4 Detector
Thermal Conductivity Detector (TCD)
Essentially universal detection and can be
used to detect any component other than
the carrier gas.
Photo Diode-Array
Monitoring more
than one absorbing
component at
a time using a large number of
diodes results in rapid analysis
and savings on expensive solvents.
6 CHROMATOGRAM
5 CHROMATOGRAM
Qualitative analysis
The chromatogram is generally represented as a graph of detector response (y-axis) vs. retention time (x-axis). This provides a spectrum of peaks representing
the analytes present in a sample eluting from the column at different times and can be used to identify complex mixtures of analytes.
Quantitative analysis
In a chromatogram, the area under the peak is proportional to the amount of analyte present. By calculating the area under the peak, the concentration of an
analyte in the original sample can be determined.
Applications
Medical Analyses
Detection of Illicit
Drugs, & Pesticides
Research
Applications
Pharma/Biophara
Manufacturing
QA/QC for
Various Products
Environmental
Assessment
Forensic
Investigation
Toxicology