2. Overview:
Chromatography and its principle
Liquid chromatography
High Performance Liquid Chromatography ( HPLC )
The components of the high performance liquid chromatograph
(HPLC).
4. The process involves the interaction of the compounds in the analyte
(which travels along with a mobile phase) across an immobile surface
(stationary phase).
The compounds bind at specific regions of stationary phase based on
certain physical and chemical properties. These bound molecules are then
eluted with a suitable buffer and the same are collected with time.
These are –
Polarity
Charge
Molecular weight
Present of functional group
Principle
5. Introduction
HPLC is a form of liquid chromatography used to separate compounds
that are dissolved in solution.
HPLC instruments consist of a reservoir of mobile phase, a pump, an
injector, a separation column, and a detector.
Compounds are separated by injecting a sample mixture onto the
column.
The different component in the mixture pass through the column at
differentiates due to differences in their partition behavior between the
mobile phase and the stationary phase.
The mobile phase must be degassed to eliminate the formation of air
bubbles.
6. Liquid chromatography is a separation technique that involves:
• the placement (injection) of a small volume of liquid sample
• into a tube packed with porous particles (stationary phase)
• where individual components of the sample are transported along the
packed tube (column) by a liquid moved by gravity.
The components of the sample are separated from one another by the column
packing that involves various chemical and/or physical interactions between
their molecules and the packing particles.
The separated components are collected at the exit of this column and
identified by an external measurement technique , such as a
spectrophotometer that measures the intensity of the color , or by another
device that can measure their amount.
7. Principles of HPLC
Principle:
The table shows relation between various parameters
of HPLC.
Trendline:
Stationary phase have small particulate size and high
surface areas.
Columns: 20 cm or less
Mobile phase pumped at high pressures of 200Bar,
3000 psi.
Flow rates: 1-3 cm3 per min
Column length No. of theoretical plates per
unit area
Resolving power Column length
Particle size Surface area
8. What is HPLC?
HPLC is a separation technique that involves:
•the injection of a small volume of liquid sample
•into a tube packed with tiny particles (3 to 5 micron ( μm ) in diameter
called the stationary phase)
•where individual components of the sample are moved down the
packed tube (column) with a liquid (mobile phase) forced through
the column by high pressure delivered by a pump.
These components are separated from one another by the column packing that
involves various chemical and/or physical interactions between their molecules
and the packing particles.
These separated components are detected at the exit of this tube (column) by a
flow-through device (detector) that measures their amount. An output from
this detector is called a “liquid chromatogram”.
10. Varian 9010 Solvent Delivery
System
Rheodyne
Injector
%A %B %C Flow Rate Pressure
{H2O} {MeOH} (mL/min) (atmos.)
Ready
Ternary Pump
A
C
B
from solvent
reservoir
Column
to
detector
to column
through
pulse
dampener
to injector
through pump
load
inject
12. COMPOSITION OF A LIQUID CHROMATOGRAPH SYSTEM
Solvent
Solvent Delivery System (Pump)
Injector
Sample
Column
Detectors
Waste Collector
Recorder (Data Collection)
13. Instrumentation of HPLC
( Describing the 5 major components and their
functions….)
1
2
3
4
5
Solvent
reservoirs
and degassing
Not shown
here
1 – Pump
2 – Injector
3 – Column
4 – Detector
5 – Computer
14. 1. Pump:
•The role of the pump is to force a liquid (called the mobile phase)
through the liquid chromatograph at a specific flow rate, expressed
in milliliters per min (mL /min).
•Normal flow rates in HPLC are in the 1-to 2-mL/min range.
•Typical pumps can reach pressures in the range of 6000-9000
psi (400-to 600-bar).
•During the chromatographic experiment, a pump can deliver a
constant mobile phase composition (isocratic) or an increasing
mobile phase composition (gradient).
15. Pump Module–types:
Isocratic pump - Delivers constant mobile phase composition;
•solvent must be pre-mixed;
•lowest cost pump
Gradient pump - Delivers variable mobile phase composition;
•can be used to mix and deliver an isocratic mobile phase or a
gradient mobile phase
–Binary gradient pump –delivers two solvents
–Quaternary gradient pump –four solvents
16.
17. 2. Injector:
•The injector serves to introduce the liquid sample into the flow stream of
the mobile phase.
•Typical sample volumes are 5-to 20-microliters (μL).
•The injector must also be able to withstand the high pressures of
the liquid system.
•An auto sampler is the automatic version for when the user has many
samples to analyze or when manual injection is not practical .
18. Sample Injection
……how is a sample actually put into an LC system
Manual Injector:
1.User manually loads sample into the injector using a syringe
2.and then turns the handle to inject sample into the flowing mobile
phase… which transports the sample into the beginning (head) of the
column, which is at high pressure
Auto sampler:
1.User loads vials filled with sample solution into the auto sampler tray
(100 samples)
2.and the auto sampler automatically
a. measures the appropriate sample volume,
b. injects the sample,
c. then flushes the injector to be ready for the next sample,
etc., until all sample vials are processed …
21. 3. Column:
• Considered the “heart of the chromatograph” the column’s stationary
phase separates the sample components of interest using various physical
and chemical parameters.
•The small particles inside the column are what cause the high
back pressure at normal flow rates.
•The pump must push hard to move the mobile phase through the
column and this resistance causes a high pressure within the
chromatograph.
22. HPLC Columns
Within the Column is where separation occurs.
Key Point –Proper choice of column is critical for success in HPLC
Materials of construction for the tubing
Stainless steel (the most popular; gives high pressure capabilities)
Glass (mostly for biomolecules)
PEEK polymer (biocompatible and chemically inert to most solvents
Packing material:
The packing material is prepared from SILICA particle, ALUMINA particle and
ion exchange RESIN.
Porous plug of stainless steel or Teflon are used in the end of the columns to
retain the packing material.
According to the mode of HPLC , they are available in different size , diameters,
pore size or they can have special materials attached ( such as antigen or
antibody ) for immuno affinity chromatography.
23. Types of columns in HPLC:
Guard Coalumn
Fast Column
Preparative(i.d. > 4.6 mm; lengths 50 –250 mm)
Capillary(i.d. 0.1 -1.0 mm; various lengths)
Nano(i.d. < 0.1 mm, or sometimes stated as < 100 μm)
Analytical[internal diameter (i.d.) 1.0 -4.6-mm; lengths 15 –250 mm]
24. Guard Column
These are placed anterior to the separating column. This serves as
protective factor.
They are dependable columns designed to filter or remove :
Particles that clog the separation column
Compounds and ions that could ultimately cause “ Baseline drift ”,
decreased resolution, decreased sensitivity and create false peaks.
These columns must be changed on a regular basis in order to
optimize their protective function.
26. Capillary Column
It is also known as micro columns
It has a diameter much less than a millimeter and there 3 types:
Open tubular
Partially packed
Tightly packed
They allow the user to work with nanoliter sample volume , decreased
flow rate and decreased solvent usage volume , led to cost effectiveness
28. Preparatory Column
Used when objective is to prepare bulk ( milligrams) of sample for
laboratory preparatory application.
It has usually a large column diameter , which is designed to facilitate large
volume injections into the HPLC system
30. 4. Detector:
• The detector can see (detect) the individual molecules that come out
(elute) from the column.
•A detector serves to measure the amount of those molecules
so that the chemist can quantitatively analyze the sample
components.
•The detector provides an output to a recorder or computer
that results in the liquid chromatogram(i.e., the graph of the
detector response).
31. UV-Vis Detectors
31
b
c
Detector Flow Cell
I0 I
Log I0 = A = abc
I
Principles: The fraction of light transmitted through the detector cell is
related to the solute concentration according to Beer’s Law.
Characteristics: Specific, Concentration Sensitive, good stability,
gradient capability.
Special: UV-Vis Spectral capability (Diode Array Technology ).
32. Electrochemical Detectors
Gold for carbohydrates.
Platinum for chlorite, sulfate, hydrazine, etc.
Carbon for phenols, amines.
Silver for chloride, bromide, cyanide.
32
34. 5. Computer:
• Frequently called the data system,
The computer not only controls all the modules of the HPLC
instrument but it takes the signal from the detector and uses it
to:
1. determine the time of elution (retention time) of the
sample components (qualitative analysis) and
2. the amount of sample ( quantitative analysis) .
37. HPLC is optimum for the separation of chemical and biological
compounds that are non-volatile .
Typical non-volatile compounds are:
Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol)
Salts like sodium chloride and potassium phosphate
Proteins like egg white or blood protein
Organic chemicals like polymers (e.g. polystyrene, polyethylene)
Heavy hydrocarbons like asphalt or motor oil
Many natural products such as ginseng, herbal medicines, plant extracts
Thermally unstable compounds such as trinitrotoluene (TNT), enzymes
HPLC used for
38. HPLC uses
This technique is used for -
chemistry and biochemistry research analyzing
complex mixtures
purifying chemical compounds
developing processes for synthesizing chemical
compounds
isolating natural products, or predicting physical
properties.
It is also used in quality control to ensure the purity of raw
materials, to control and improve process yields, to quantify assays
of final products, or to evaluate product stability and monitor
degradation.
