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Separation Science

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HPLC

Used to separate, identify, and quantify


compounds based on the interactions between
the stationary phase, the sample, and the
solvents used.

GC

vs.

Used to separate and analyze compounds


that can be vaporized without decomposition.

Organic Molecules, Biomolucules, Ions, & Polymers

Organic or Inorganic Compounds. Must be volatile,


salt-free, and should not contain ions.

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

Pump performance is measured


on their ability to yield a consistent
and reproducible flow rate.

Ar

Common pump types include:


Constant Pressure Pumps Provide
constant flow through the column
with the use of pressure from a gas
cylinder.

Syringe Pump Suitable for small bore


columns, constant flow is delivered by a
motorized screw arrangement.
Reciprocating Piston Pump Deliver solvents
through reciprocating motion of a piston in a
hydraulic chamber.

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.

On-column Injection This is the method


of choice for all samples with high-boiling
point components that would not be
transferred on split or splitless injection.

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

Common column types include


normal and reverse phase columns,
ion exchange columns, size exclusion
columns, and chiral columns.

Column choice depends on the sample and


active measured. The main attribute to
consider is polarity, but functional groups
can also affect column selection. To increase
separation and decrease run time, the
polarity of the column should closely match
the polarity of the stationary phase. Film
thickness, column diameter, and length also
affect run time.
Gas chromatographic columns are usually
between 1 and 100 meters long.

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.

Flame Ionization Detector (FID) Robust


and sensitive to hydrocarbons but cannot
detect water.
Selective detectors that only respond to
particular substances include:




Mass Spectroscopy Detection


based on fragmentation of
molecules by electric fields and
separation by mass ratio offers
very high selectivity and sensitivity.

6 CHROMATOGRAM

Mass Spectrometer (GC/MS)


Electron Capture Detector (ECD)
Nitrogen-phosphorous
Flame Photometric (FPD)
Photo-ionization (PID)

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