3. HPLC- HISTORICAL PERSPECTIVE AND THE NAME
• Better separation
• Quicker separation time
• Easier Analysis
• Better Results
HIGH
PERFORMANCE
1903
1952
Mikhail Tsvet, a botanist from
Russia, invented chromatography
Archer John Porter Martin, & Richard
Laurence Millington Synge - The Nobel
Prize in Chemistry in 1952 for their
invention of Partition Chromatography
6. 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.
7. 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.
8. 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.
9. 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.
10. 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.
11. SAMPLE INJECTOR:
1) RHEODYNE OR LOOP SAMPLE INJECTOR:
• Manual sample injector introduced by Rheodyne
corporation.
• Has six-port valve system and two positions.
• First position is load position and second position is
inject position.
• 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.
2) SEPTUM INJECTOR:
• Consists of a rubber septum through which a
needle is inserted to inject the sample.
• Septum acts as a seal of an injector port.
• Septum must withstand high pressure generated
in the system.
12. STOP FLOW INJECTOR
• In this type of injector, flow of the mobile
phase stops when a sample is injected.
• Due to the mechanism of stop flow, a
ghost peak is generated in this type of
injector
AUTO SAMPLER
FEATURES PUSH-LOOP DESIGN PULLED-LOOP
DESIGN
SPLIT-
LOOP,INTEGRATED-
LOOP, FLOW-
THROUGH NEEDLE
MECHANICAL
SIMPLICITY
Very simple design Moderate
complexity
Complex
EASE OF FLUSHING No Very easy to flush Very easy to flush
CARRYOVER Low Low Very low
IMPACT ON DWELL
VOLUME
Less Medium Less
CONSERVES
SAMPLE/CONSUMPT
ION OF SAMPLE
Low conservation,
high consumption
Medium
conservation,
medium
consumption
High conservation,
low consumption
INJECTION
FLEXIBILITY
Low flexibility High flexibility Very high flexibility
NEEDLE SEAL
PROBLEMS/
PRECISION OF
INJECTION VOLUME
Less issues Moderate issues High issues
13.
14. HPLC COLUMN CLASSIFICATION BASED ON SCALE OF USE
COLUMN CLASSIFICATION
BASED ON SCALE OF
OPERATION
SCALE AND OBJECTIVE OF
COLUMNS
COLUMN DIAMETER PARTICLE SIZO OF
CLOUMN
ANALYTICAL For identification and
quantitative analysis
1 - 8 mm 1.7 – 10 micron
SEMI-PREPARATIVE Purification of compound
with less than 0.5 gram
10 – 40 mm 5 – 15 micron
PREPARATIVE Compounds can be
purified up to more than
0.5g but less than kg.
50 – 100 mm 15 – 100 micron
PROCESS/INDUSTRIAL
PRODUCTION
Manufacturing quantity
from grams to kg
More than 100 mm More than 100 micron
15. SEPARATION BASED ON
POLARITY/CHARGE
CATION EXCHANGE CHROMATOGRAPHY:
• Retains & separates +ve ions on a -ve surface
ANION ECHANGE CHROMATOGRAHY
• Retains & separates –ve ion on a positive surface
SIZE EXCLUSION/GEL PERMEATION/GEL
FILTRATION
• Smaller molecule of the compound in the solution
diffuses deeper into stationary phase matrix
• Thus, smaller molecule elution is impaired while larger
molecules elute faster.
16. COLUMN SELECTION/CLASSIFICATION
BASED ON PHYSICAL/CHEMICAL
ATTRIBUTES
SIGNIFICANCE OF PORE SIZE OF STATIONARY
PHASE AND MOLECULAR WEIGHT
PORE SIZE OF PAXCKING
MATERIAL
MOLECULAR WEIGHTS IN
DALTONS/KDa
80 - 120 Up to a molecular weight of
2000
200 - 450 Over 2000
1000 & 4000 Very high molecular weight
proteins and vaccines
17. 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
19. DATA ACQUISITION MODULE
• Interface between a machine and a
user.
• Used to program and command the
HPLC, read and interpret the data
and store the acquired data
WORKSTATION TO PROCESS REQUIRED DATA
• Consists of two components viz.
a) Data acquisition:
• Acquires signal from detector and convert analog signals
to digital
b) Data processing:
• 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
20. APPLICATIONS
OF HPLC
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 :
• Phenol & mercury from seawater
3. Forensic Test :
• 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
21. HPLC CALIBRATION PARAMETERS AND RECOMMENDED FREQUENCY
QUATERLY PARAMETERS HALF-YEARLY PARAMETERS YEARLY PARAMETERS
Pressure test UV-VIS/PDA detector by linearity
measurement
RI detector by linearity
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
measurement
UV-VIS/PDA detector by wavelength
accuracy measurement
Pump by gradient flow
measurement/
UV-VIS/PDA detector by reference
energy check
Autosampler for RI detector by
linearity measurement
