HPLC vs gcUsed to separate, identify, and quantify

compounds based on the interactions

between the stationary phase, the sample,
and the solvents used.
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. 1 gas supply
Additives such as salts
Typical carrier gases include
and acids may also
helium, nitrogen, argon,
be included for some
hydrogen, and air and are
techniques.
usually determined by
He the detector being used.
Higher flow rates yield faster

Pump 2 analysis, but less separation

between analytes.
Pump performance is measured N
on the instrument's ability to yield a
consistent and reproducible flow rate.
Constant Pressure Pumps – Provide
constant flow through the column Ar
with the use of pressure from a gas
cylinder.
Syringe Pump – Suitable for small bore
columns, constant flow is delivered by a
H
motorized screw arrangement.
Reciprocating Piston Pump – Deliver solvents
through the reciprocating motion of a piston
in a hydraulic chamber.

2 sample injector
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.
3 On-column Injection – This is the method
of choice for all samples with high-boiling
AUTOSAMPLER point components that would not be
transferred on split or splitless injection.
Autosamplers ensure reliable, precise, and
accurate injection and support a wide range of
formats and sample throughputs.
Over 95 percent 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.
Common column types include
3 Capillary column
normal and reverse phase columns, Column choice depends on the sample and
ion exchange columns, size exclusion active measured. The main attribute to
columns, and chiral columns. 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.

5 Detector
UV-Vis – Its oven
response is specific
to a particular
compound
depending on
the presence of
light absorbing
functional groups. 4 Detector
Photo Diode-Array
Thermal Conductivity Detector (TCD) –
– Monitors multiple
Essentially universal detection and can be
components at
used to detect any component other than
a time using a
the carrier gas.
large number of
diodes, resulting Flame Ionization Detector (FID) – Robust
in rapid analysis and sensitive to hydrocarbons but cannot
and savings on detect water.
expensive solvents.
Selective detectors:
– Detection based on
• Mass Spectrometer (GC/MS)
fragmentation of molecules by
• Electron Capture Detector (ECD)
electric fields and separation by
• Nitrogen-phosphorous
mass ratio offers
• Flame Photometric (FPD)
very high selectivity and sensitivity.
• Photo-ionization (PID)

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 Research Pharma/Biophara QA/QC for Environmental Forensic Toxicology
Drugs & Pesticides Applications Manufacturing Various Products Assessment Investigation