INTRODUCTION TO HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
WHAT IS HPCL?
It is a process of separating components in a liquid mixture.
PRINCIPLE: A liquid sample is injected into a stream of solvent (Mobile Phase) flowing through a column
with a separating medium (Stationary Phase).
It is suitable for both volatile and non-volatile compounds.
Solubility is important in HPCL since compounds must be soluble in the mobile phase.
Less destructive nature whereas, samples can often be recovered after.
HOW HPCL WORKS?
TWO PHASES:
MOBILE PHASE (MOVABLE)
- Involved in the movement of compounds through the column.
- It is in a liquid state, usually a solvent or a mixture of solvents.
- Polarity can be adjusted by mixing different solvents.
EXAMPLES OF SOLVENTS:
a) Polar solvents: water, alcohols (mobilize polar compounds)
b) Mid-Range Polarity: Ethyl Acetate, methylene chloride.
c) Non-Polar Solvents: Hexanes, ethers (mobilize non-polar compounds)
STATIONARY PHASE
- No movement; retains or adheres compound.
- It is in a solid chemical phase and bound within a column.
*A column is a long tube, and it is coated with material (e.g., C18, C8, Ion Exchange). It is responsible for binding
compounds.
Simple Process of HPCL:
1. The sample is loaded into the column with the mobile phase.
2. Sample adheres or sticks to the stationary phase
3. The mobile phase is pumped through at a steady rate.
4. Compounds are washed along based on their affinity for the stationary and mobile phases.
SIMPLE DIAGRAM
MOBILE PHASE
CHROMATOGRAPH
STATIONARY DETECTO
PHASE R
MODES OF HPLC
1. NORMAL PHASE HPLC
- Column Polarity: Non-Polar (E.G C18 COLUMN)
- Mobile Phase Polarity: Polar
2. REVERSE PHASE HPLC:
- Column Polarity: Polar
- Mobile Phase Polarity: Non-polar (e.g. methanol, water, acetonitrile)
3. ION-PAIR CHROMATOGRAPHY
4. ION EXCHANGE CHROMATOGRAPHY
- Mobile Phase: Often uses a buffer (such as sodium).
5. SIZE EXCLUSION CHROMATOGRAPHY
HPLC INTSRUMENT COMPONENTS
PARTS OF THE HPLC:
1. Mobile Phase Reservoirs: Holds the solvents or
buffer solutions.
2. Degasser- Removes the air bubbles from the
mobile phase to ensure a stable baseline.
3. Pumps- Deliver the mobile phase at a constant
and controlled flow rate (adjustable; affects
retention time)
4. Auto-sampler/injector- Introduces the sample
into the mobile phase stream.
5. Column (Stationary)- Where the separation
occurs.
6. Detector: Detects the separated compounds as
they elute from the column.
7. Data System/Software- Collects and analyzes the
detector signal to generate a chromatograph.
CHROMATOGRAPH INTERPRETATION:
DEFINITION OF TERMS:
Y-AXIS: Detector Response (Abundance)
X-AXIS: Elution Time (minutes)
BASELINE: Must be stable for accurate analysis.
PEAKS: Represents the individual compounds.
T0 (T NAUGHT): Dead time; elution time for unretained material. It is usually observed at the beginning
of the graph.
Retention Time (T.R): Time it takes for a compound to elute from the column.
Peak Area: Proportional to the amount of the analyte.
Calibration Curve: It is used to quantify the amount of analyte based on the peak area.
