HIGH

PERFORMANCE
LIQUID
CHROMATOGRAP
HY
BY,
Abhijit Padhi
HPLC- HISTORICAL PERSPECTIVE AND THE
NAME

HIGH
PERFORMANCE

1903
Mikhail Tsvet, a botanist from
• Better separation
Russia, invented chromatography
• Quicker separation time
• Easier Analysis
1952 • Better Results

Archer John Porter Martin, & Richard

Laurence Millington Synge - The Nobel
Prize in Chemistry in 1952 for their
invention of Partition Chromatography
 PAPER
CHROMATOGRAPHY

GAS THIN LAYER

CHROMATOGRAPHY CHROMATOGRAPHY

TYPES OF
CHROMATOGRAP HPLC

HY AFFINITY GEL PERMEATION

CHROMATOGRAPHY CHROMATOGRAPHY

ION EXCHANGE
CHROMATOGRAPHY
 SOLVENT RESERVOIR
• Made up of glass covered with special caps.
• Holds the mobile phase or solvent.
• Pumped through the system with the help of
mobile phase transfer line & high-pressure
pump.
MOBILE PHASE TRANSFER LINE
• Tubing with long length and small diameter.
• Stainless steel/Polyether ether ketone (PEEK).
• Pump mobile phase through the HPLC system.
• Operating pressure : 5800-7000 psi.
• For UPLC : up to 15000 psi.
 FRITS (Sinker frits in the mobile
phase reservoirs)
• Sinker frits (0.5 micron – 10 micron pore size)
attached at the end of inlet tubing that dips
into the mobile phase reservoir.
• Provides protection against particulate
contaminants entering the pump, injector and
column.
• Helps keep the inlet tubing submerged in the
mobile phase.
DEGASSING SYSTEM
• Dissolved gases are removed from the
solvents by applying vacuum to a semi -
permeable membrane.
• High efficiency Teflon material allows the
usage of a very short length of capillary inside
the vacuum chamber.
HIGH PRESSURE HPLC
PUMP:
Maintain a constant flow of mobile phase regardless of resistance & back pressure because of column packing.

1. Constant Pressure Pump :

Pressure is generated using a gas cylinder.

• Provides pulse-less and continuous pressure with high flow rates.
• Not suitable for gradient elution.
2. Constant Flow Pump:
Based on two basic principles:
a) Positive displacement (syringe pump):
• Useful for precise constant flow without pulsation
where there is a constant load.
• Can also be used to generate flow by two or multiple
syringes.
• Mostly used for micro or nano HPLC instruments.
Disadvantage : Requires frequent filling
b) Reciprocating Piston Pump:
Pumping process is driven by a stepper motor.
• Motor drives a rotating disc or cam that pulls the piston back
& forth.
• A small amount of mobile phase is pumped, during each
pump stroke.
• A flexible flushing seal in the piston, prevent solvent leakage
from the pump.
• Check valves maintain pressure & a one-way mobile phase.
• Classified into single, dual pistons in-parallel and in-series
types.
3. HIGH-PRESSURE PUMP
• Consists of two or three pressure pumps for conveying
different solvents.
• Separate pumps deliver solvent to the mixing chamber.
• Mixing of solvent occurs with high pressure (at the high
pressure side)
• Therefore, such a design is called high-pressure gradient
system.
• Comparatively more expensive than low-pressure system. •
• Precise, reproducible, and better composition accuracy up
to 0.1%.
• Lower dwell times.
4. LOW-PRESSURE PUMP
• Consists of two or more mobile-phase reservoirs connected
with a solenoid valve, which is further connected with a
mixing chamber.
• Valves can be controlled to provide desired composition of
the mobile phased in the mixing chamber.
• Mixing of mobile phase occurts on the low-pressure side
prior to entering the pump.
• Less expensive
Capable of providing quaternary systems for operation.
• Demerits are: Higher dwell volume.
• Degassing system id necessary
• Less compositional accuracy hence less retention time
precision.
SAMPLE INJECTOR:
1) RHEODYNE OR LOOP SAMPLE INJECTOR: 2) SEPTUM INJECTOR:

• Manual sample injector introduced by Rheodyne • Consists of a rubber septum through which a
corporation. needle is inserted to inject the sample.
• Has six-port valve system and two positions. • Septum acts as a seal of an injector port.
• First position is load position and second position is • Septum must withstand high pressure generated
inject position. in the system.
• Sample volume is interchangeable.
• A 22 gauge needle with a blunt tip, is used to inject the
sample manually.
• Once the sample is injected at load position, the injector
is manually rotated to set the injection position.
• It does not create air bubbles.
• Does not disturb the system pressure and flow rate.
 AUTO
STOP FLOW INJECTOR SAMPLER
FEATURES PUSH-LOOP DESIGN PULLED-LOOP SPLIT-
DESIGN LOOP,INTEGRATED-
LOOP, FLOW-
THROUGH NEEDLE

MECHANICAL Very simple design Moderate Complex

SIMPLICITY complexity

EASE OF FLUSHING No Very easy to flush Very easy to flush

CARRYOVER Low Low Very low

• In this type of injector, flow of the mobile IMPACT ON DWELL

VOLUME
Less Medium Less

phase stops when a sample is injected.

CONSERVES Low conservation, Medium High conservation,
• Due to the mechanism of stop flow, a SAMPLE/CONSUMPT high consumption conservation, low consumption
ION OF SAMPLE medium
ghost peak is generated in this type of consumption
injector
INJECTION Low flexibility High flexibility Very high flexibility
FLEXIBILITY

NEEDLE SEAL Less issues Moderate issues High issues

PROBLEMS/
PRECISION OF
INJECTION VOLUME
 HPLC COLUMN CLASSIFICATION BASED ON SCALE OF USE
COLUMN CLASSIFICATION SCALE AND OBJECTIVE OF COLUMN DIAMETER PARTICLE SIZO OF
BASED ON SCALE OF COLUMNS CLOUMN
OPERATION

ANALYTICAL For identification and 1 - 8 mm 1.7 – 10 micron

quantitative analysis

SEMI-PREPARATIVE Purification of compound 10 – 40 mm 5 – 15 micron

with less than 0.5 gram

PREPARATIVE Compounds can be 50 – 100 mm 15 – 100 micron

purified up to more than
0.5g but less than kg.

PROCESS/INDUSTRIAL Manufacturing quantity More than 100 mm More than 100 micron
PRODUCTION from grams to kg
 SEPARATION BASED ON SIZE EXCLUSION/GEL
POLARITY/CHARGE PERMEATION/GEL FILTRATION

 CATION EXCHANGE CHROMATOGRAPHY: • Smaller molecule of the compound in the solution

• Retains & separates +ve ions on a -ve surface diffuses deeper into stationary phase matrix
• Thus, smaller molecule elution is impaired while larger
molecules elute faster.
 ANION ECHANGE CHROMATOGRAHY
• Retains & separates –ve ion on a positive surface
 SIGNIFICANCE OF PORE SIZE OF
COLUMN SELECTION/CLASSIFICATION STATIONARY PHASE AND MOLECULAR
BASED ON PHYSICAL/CHEMICAL WEIGHT
ATTRIBUTES

PORE SIZE OF PAXCKING MOLECULAR WEIGHTS IN

MATERIAL DALTONS/KDa

80 - 120 Up to a molecular weight of

2000

200 - 450 Over 2000

1000 & 4000 Very high molecular weight

proteins and vaccines
 SIGNIFICANCE OF PARTICLE SIZE OF STATIONARY
PHASE

• Smaller the particle size of packing material

in the column, higher will be the efficiency
and higher backpressure.

• When the particle size of a column is

decreased by half the plate
number/theoretical plate count doubles
(when column length and internal diameter
of the column remains the same in both
cases) and column backpressure increases
by four times
HPLC DETECTION SYSTEM
 WORKSTATION TO PROCESS
DATA ACQUISITION MODULE REQUIRED DATA

• Consists of two components viz.

• Interface between a machine and a
a) Data acquisition: user.
• Acquires signal from detector and convert analog • Used to program and command the
signals to digital HPLC, read and interpret the data
b) Data processing: and store the acquired data
• Further process signals from detector and, compute in
numerical form and provides graphical representation
of the data in the form of Chromatogram, that is easy
to read, understand and interpret
 1. Pharmaceutical industry :
• To control the drug stability
• Quantitative estimation of drug from pharmaceutical dosage forms eg. Paracetamol
determination in Panadol tablet
• Quantitative estimation of drug sample from biological fluids eg. blood glucose level.
2. Analysis of natural contamination :
APPLICATIO 3.
• Phenol & mercury from seawater
Forensic Test :
NS OF HPLC • Determination of steroid in blood, urine & sweat
• Determination of psychotropic drug in plasma
4. Clinical Test :
• To check for metabolites produced in the body, Vitamin D analysis, Hb1Ac determination,
immunoassay and enzymatic assay.
5. Food & essence manufacturing :
• Sweetener analysis in fruit juice, preservative analysis in sausage
 HPLC CALIBRATION PARAMETERS AND
RECOMMENDED FREQUENCY
QUATERLY PARAMETERS HALF-YEARLY PARAMETERS YEARLY PARAMETERS
Pressure test UV-VIS/PDA detector by linearity RI detector by linearity
measurement measurement

Drift and Noise Auto sampler by carry over check Fluorescence detector by linearity
measurement

Column oven and sample cooler Auto sampler by linearity check Fluorescence detector by
wavelength accuracy measurement

Pump by flow rate accuracy UV-VIS/PDA detector by wavelength

measurement accuracy measurement

Pump by gradient flow Autosampler for RI detector by

measurement/ linearity measurement
UV-VIS/PDA detector by reference
energy check
 QUERIES??

THANKS FOR
YOUR PATIENCE