HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Chromatography – is a multistage separation method

- Components:
 Stationary Phase - may be a solid or a liquid supported on a solid or a gel;
may be packed in a column, spread as a layer, distributed as a film, or applied
by other techniques
 Mobile Phase - may be in a gaseous or liquid form, or a supercritical fluid

- Separation may be based on:

 Adsorption
 Mass distribution (partition)
 Ion exchange
 Differences among the physicochemical properties (size, mass, and volume) of
the molecules

Liquid Chromatography
- is a separation technique based on a solid stationary phase and a liquid mobile
phase.
- Apparatus: A liquid chromatograph consisting of
 a reservoir containing the mobile phase
 a pump to force the mobile phase through the system at high pressure
 an injector to introduce the sample into the mobile phase
 a chromatographic column
 a detector
 a data collection device
SHIMADZU LIQUID CHROMATOGRAPH
HITACHI LIQUID CHROMATOGRAPH
AGILENT LIQUID CHROMATOGRAPH
Common parts of an HPLC apparatus:
 Pump – determines the pressure at which the mobile phase has to be pumped into the
system
- Adjusts the flow rate depending on the substance to be tested

 Injector – introduces the liquid sample into the flow stream of the mobile phase

 Column – separates the desired component from the pool of samples using various
physical and chemical parameters
- comprises of a metal housing in a tube shape which is packed with tiny beads that have
an affinity for the chemicals that are being analyzed

 Detector – detects individual molecules that come out of the column

- Functions by having set a specific wavelength or using a specific lamp that can detect
the molecule desired

 Communication bus model - connects the system to the computer

- Gathers the data collected by the system and relays it to the computer for analysis and
interpretation

Optional Parts:
 Degasser – removes air bubbles that may alter the results

 Oven – for components that can only be separated at temperature above or below
normal room temperature

Schematic diagram:
When using HPLC system:
- Check if the apparatus is clean, calibrated, and functioning properly
- Assure that there are no traces of the precious analysis in the system
- Ensure that the mobile phase is flowing through the flow line properly and that
there are no leaks observed anywhere

When preparing the mobile phase:

- Always prepare fresh, prior before use
- Check pH
- Degas so that there are no bubbles or any impurities that may alter the results
- Place a label including the product name, preparation procedure, reference,
name of the person who prepared it, date prepared, and expiration date

 Preventive maintenance: done quarterly (every three months) by removing the

system’s parts and cleaning them individually to ensure cleanliness and that they are
functioning properly so that they can last a long time.

 Calibration: done quarterly (every three months) after preventive maintenance to

check if the functioning of the components of the system is within the standards set.