HPLC
HPLC
HPLC
INSTRUMENTATION
Lect. Delivered:
Dr Ali Imran Khawaja .
Operation:-
A further refinement to HPLC has been to vary the mobile phase composition during the
analysis; this is known as gradient elution. A normal gradient for reversed phase
chromatography might start at 5 % methanol and progress linearly to 50 % methanol
over 25 minutes, depending on how hydrophobic the analyte is. The gradient separates
the analyte mixtures as a function of the affinity of the analyte for the current mobile
phase composition relative to the stationary phase. This partitioning process is similar to
that which occurs during a liquid-liquid extraction but is continuous, not step-wise. In
this example, using a water/methanol gradient, the more hydrophobic components will
elute (come off the column) under conditions of relatively high methanol; whereas the
more hydrophilic compounds will elute under conditions of relatively low methanol.
The choice of solvents, additives and gradient depend on the nature of the stationary
phase and the analyte. Often a series of tests are performed on the analyte and a number
of generic runs may be processed in order to find the optimum HPLC method for the
analyte - the method which gives the best separation of peaks.
Flow chart of HPLC:
Types of HPLC:-
1. Normal Phase HPLC.
2. Reverse Phase HPLC.
3. Size Exclusion HPLC.
4. Ion- exchange HPLC.
1. Normal Phase HPLC:-
“In the normal phase HPLC the stationary is a polar adsorbent and the
mobile phase is generally a mixture of non-aqueous solvents”.
Normal phase HPLC was the first kind of HPLC chemistry used, and it separates
analyte based on polarity. This method uses a polar stationary phase and a non-polar
mobile phase, and used when the analyte of interest is polar in nature.
Principle:-
“Adsorption of analyte on the polar, mostly weakly acidic surface of silica gel”.
The polar analyte associates with and is retained by the polar stationary
phase. Adsorption strength increases with increase in analyte polarity and the interaction
between polar analyte and stationary phase, thus increases the elution time. The
interaction strength not only depends upon the functional groups in the analyte
molecule, but also on steric factors and structural isomers that are often resolved from
one another.
STRUCTURAL FORMULA:
Use of more polar solvents in the mobile phase will decrease the retention time
of analyte while more hydrophobic solvents tend to increase the retention time.
Stationary Phase:-
Stationary phases commonly used in normal phase HPLC include solid adsorbents
such as.
SiO2
Al2O3
NH2
CN
NO2
Bonded Diols
Mobile phase:-
Mobile phases commonly used in NP – HPLC include:
Heptanes.
Hexane.
Cyclohexane.
CHCl3
Mechanism in Normal Phase HPLC:-
The sample bind to polar sites of stationary phase, it is a physical attachment and
a dynamic reversible process. The sample molecules that had been adsorbed on
stationary phase should be detached to elute out from the column. In order to carry out
this process we use different types of mobile phase having different polarity index.
Forces that play role in N.P HPLC:
Dipole - Dipole (Normal Phase)
Intermolecular forces resulting from the attraction of the positive and negative ends of
the dipole moments of polar molecules
Dipole - Induced Dipole (Normal Phase)
Even if a molecule has no dipole moment a temporary dipole moment can be induced
when one molecule with a dipole approaches another molecule in which the electrons
are slightly displaced from a symmetrical arrangement.
Particularly polar solvents in the mixture tend to deactivate the column by
occupying stationary phase surface; this causes detachment of sample molecules from
stationary phase and causes elution. This binding of mobile phase to the stationary phase
is independent of the sample molecules which have been already attached to the
stationary phase.
This is somewhat particular to normal phase because it is purely an adsorptive.
Mechanism:-
Separation in SE-HPLC requires carful matching of the pore size of the stationary
phase material with the size of molecules to be separated. Small molecules in a sample
will be able to penetrate all the pores of stationary phase and will elute early. Very large
molecules will be excluded from all the pores of stationary phase and will elute with the
least speed. Molecules of intermediate size will be able to penetrate some but not all of
the pores and will elute with intermediate speed.
Unlike the other modes of HPLC, interactions with the stationary phase surface
are to be avoided in SE-HPLC.
Stationary Phase:-
Stationary phases for SE-HPLC can be conveniently divided into.
Rigid polymers Silica gel, derivatized silica gel and polystyrene.
Soft gels cross linked dextran (Sephadex).
Mobile Phase:-
Any depending upon the type of the sample.
Applications:-
It is also useful for determining the tertiary structure and quaternary
structure of purified proteins. Because the gentle elution conditions tend not
to denature the analytes.
This technique is widely used for the molecular weight determination of
polysaccharides.
SE-HPLC is extensively used for the preparative separations of
macromolecules of biological origin.
Disadvantages:-
Principle:-
Stationary Phase:-
Cationic exchangers.
Anionic exchangers.
Cationic Exchangers:-
These exchangers retain the negatively charged components of analyte e.g.
sulphonic acids (strong exchanger that are ionized over a complete pH range),
carboxylic acids (weak exchangers that are ionized over a certain pH value).
Anionic Exchangers:-
These exchangers retain the positively charged components of analyte e.g.
quaternary ammonium ions (strong exchanger), amines (weak exchanger).
These ion exchangers are covalently attached to either silica gel, cellulose or
dextrans and polystyrene resins. These polymers make ion exchanger resistant to
chemical degradation and give high mechanical strength.
Mechanism:-
Retention IE-HPLC is determined by the pH of mobile phase, ionic strength,
temp and the nature and the concentration of buffer ions in the mobile phase. Organic
modifiers such as methanol and acetonitrile may also be added to the mobile phase
retention of hydrophobic ions may also arise as a result of interactions with the S.P
support or functional groups of hydrocarbons.
The ability of ion exchangers to distinguish ions of different charge is high and the
separation of ions of different charges is generally conducted under gradient elution
conditions.
An increase in counter ion (with respect to the functional groups in resins)
concentration reduces the retention time.
An increase in pH reduces the retention time in cation exchange while a
decrease in pH reduces the retention time in anion exchange.
Applications:-
o purifying water,
o preconcentration of trace components,
o Ligand-exchange chromatography,
o Ion-exchange chromatography of proteins,
o High-pH .anion-exchange chromatography of carbohydrates and
oligosaccharides, etc.
Components of HPLC:-
1. Solvent Reservoir:- It is the chamber containing the solvent used as
mobile phase. A modern HPLC is equipped with one or more glass or
stainless steal reservior, each of which contains 0.5 – 3 liters of the
solvent.
2. Mixing chamber:- It helps in the mixing of solvents according to correct
ratio.
3. De-gassing chamber: - It is the chamber in which de-gassing of mobile
phase takes place i.e. air bubbles are removed from the solvent. Argon &
Helium are used for this purpose.
4. Pump:- As a consequence of large back pressure encountered due to
small particle size of packing used in HPLC column. Pumps must be
employed to achieve acceptable eluent flow rates. They are constructed
from materials which are resistant to organic solvents and aqueous
buffering solutions commonly used as eluent.
5. Guard Column:- It is protective in its function, act like filter to remove
the impurities.
6. Injection Port:- The port from where we inject the sample under high
pressure with the help of Hypodermic syringe.
7. Analytical Column:- it is the column used for the separation of different
components of the analyte over the basis of interaction with mobile phase
and stationary phase.
8. Detectors:- It is convenient to use continuous monitoring detectors
located at the column exit. They are used to detect incoming components
from column. In HPLC there are different types of detectors. e.g. U.V
detector, Florescence detector, Refractive index detector.
9. Data Collector:- They collect the data coming from detector and sent to
recorder.
10. Recorder:- It records the reading into chromatogram.
11. Chromatogram:- It is graphical presentation of data.
Flow splitter:- It gives continuously flow rate. It measures the amount of flow of liquid
passing through column. Flow rate is measured in ml/min.
OVER VIEW OF HPLC
1. Solvent Reservoir:-
The source or reservior of liquid used in HPLC is called solvent reservior and
the mobile phase refers to the solvent being continuously applied to the column. In the
solvent reservior we have the rubber tubing pass in the bottle which are under software
control.
Mobile phase consist of mixture of organic solvents or buffering agents. These
may be employed depending upon the chromatographic method and detector to be used.
Special grades of solvents are commercially available for HPLC that have be
adequately refined to eliminate competitively the U.V absorbing impurities ( traces of
water and ethanol) and any particular matter that influence the separation.
Solvent reservoirs have the capacity of 500 ml, 1 liter, 2 liter and 3 liter. On the
basis solvent there are two methods to run out HPLC.
1. Isocratic elution.
2. Gradient elution.
Composition of Solvent used in HPLC:-
Organic solvent.
Aqueous solvent.
Buffering agent.
Polarity Index:
Affinity to measure the degree of affiliation of any solvent with polar solute.
Table:
Seria Solvent Polarity Elution U.V
l No. Index Strength Transmission
(SiO2)
1. Water 10.2 Large 170
2. Nitro methane 6.0 0.49 380
3. Acetonitrile 5.8 0.50 190
4. Methanol 5.1 0.73 205
5. Acetic acid 4.4 0.38 255
6. Chloroform 4.1 0.26 235
7. Tetra hydro 4.0 0.35 210
furan
8. Dichloromethane 3.1 0.34 230
9. Carbon 1.6 0.11 265
tetrachloride
2. 10. N- Hexane 0.1 0.01 195
11. Cyclohexane 0.04 0.03 200
Degassing Chamber:-
Many liquids dissolve appreciable amount of atmospheric gases e.g. air or suspended air
bubbles that may be a major cause of practical problems in HPLC, specifically affecting
the operation of pumps and the detectors.
However all such problems may be avoided by degassing the mobile
phase by subjecting the mobile phase under vacuum distillation ,
spraying with affine spray of an inert gas of low solubility such as Ar
or He and by heating.
1. Pumps:-
The pump can be thought of as the heart of HPLC system, the instrument
that makes the mobile phase at the first glance, the attributes required of the HPLC
pumps are simple: it should be able to deliver the mobile phase through the column at a
constant and reproducible flow rate and be able to do this in a pulse free manner.
The Ideal HPLC Pump:-
To design an ideal HPLC pump, the following features should be
built into it.
1. Pressure Generation: the pump should be able to generate a high
pressure associated with modern HPLC columns and cope with these
over long periods of time. It should certainly have some means of
indicating these pressure to outside the world.
“ the pressure experienced by the pump as it forces mobile phase through the column
known as column back pressure”.
They must be capable enough to create pressure of 6000 psi.
Pressure inside the HPLC is 10,000 psi.
2. Flow Rate: the flow rate from the pump should be accurate, regardless
of the system in which pump is used. This means that the flow rate
actually produced by pump is used, the same as that dialed upon the front
panel and that this should not be affected by the rest of the HPLC system.
Pulsative flow rate can limit the sensitivity of HPLC assays, resulting in
a rhythmic variation in the apparent refractive index of the mobile phase
flowing through detectors, which ultimately manifests itself in the
chromatogram as base line noise. So the flow rate should be reproducible
and practically free of pulsation.
flow rate should be 0.1 – 10 ml/min.
ideal pump should be able to provide a wide range of flow rates.
3. Resistance: the pump should be inert to the various solvents, buffer salts
and solutes to which it will be exposed in general use. In vast majority of
applications stainless steal is used for metallic parts that contact mobile
phase and this is perfectly acceptable.
Types Of HPLC Pumps:-
There are several different types of HPLC pumps available. The two main
classes are.
Constant Flow Pump.
Constant Pressure Pump.
1. Constant Flow Pump:-
They maintain constant flow of liquid, pressure can be varied. It is further
divided into two types:
Reciprocating pump.
Syringe pump.
2. Constant Pressure Pump:-
They maintain constant pressure of liquid. it is further divided as two types:
Pressurized coil pump.
Pressurized intensifier pump.
Reciprocating Pump. Syringe Pump.
80 – 90% use. 10 – 20% use.
Solvent holding capacity is Solvent holding capacity is
more than 250 ml. up to 250 ml.
Easily controllable flow Constant flow rate.
rate. Pulse free output.
Pulsative output
1. Syringe pump:
The construction of this pump is similar to the constant pressure pump but the difference
is that it has screw driven piston and digital stopper motor. Its principle is similar to that
of syringe(plunger). When piston moves upward inlet valve is closed and outlet is open,
piston pushes the solvent towards the column and vice versa.
Diagram:
4. Injection port
In order to obtain maximum chromatographic efficiency the
sample to be analyzed should be introduced on to the head of the column as an
extremely narrow band.
Loading position has no interference with the mobile phase, it just adjust the
amount of sample. The loop is filled with sample from a syringe when the valve is in the
load position. We adjust the sample loop according to the required amount of sample.
We inject large amount of the sample in the sample loop, required amount will remain
in the loop and extra amount will be drain out.
In this case sample loop is not connected with solvent reservior but sample inlet
and outlet are connected with sample loop.
Some valves require relatively large volume of sample to work efficiently, this is
particularly the case with external loop valves. It is easily adaptive for automatic
operation.
Inject Position:
Diagram:
By moving the knob we adjust the load position and the inject position. When
remove the knob to inject position then there is no connection between sample in and
sample out. And the required amount, which was filled in the loading position, will
move to the column with mobile phase coming from the pump. This system is simple
and reliable to use and has the advantage that the extra column volume due to
connecting tubing is minimized.
In this case sample loop is connected with solvent reservior but sample inlet and
outlet are not connected with sample loop.
Disadvantage:-
The disadvantage of internal loop is that they are fixed and can only be altered
by changing the roller.
5. Columns:-
Column is that area where separation takes place
There are various types of column:
1. Guard column.
2. Analytical column.
3. Derivatizing column.
4. Micro column.
5. Fast column.
6. Preparative column.
1. Guard column:-
They are installed just before the analytical column.
There length up to 10 cm.
They provide the protection to the analytical column.
They remove any foreign particles (that cause the precipitation upon
contact with stationary phase thus reducing the resolution) present in the
mobile phase and increase the shelf life of analytical column.
They act like filter.
They remove those substances that get adsorbed to the analytical column.
The composition of the guard column remain same as that of analytical
column but difference is that separation does not take place in guard column because
they are not density packed.
2. Analytical column:-
Normally these columns are made up of stain less steal 90%.
Occasionally they made up of glass 5%.
The length of the column is 10 – 30 cm.
The internal diameter is 4 – 10 mm.
Packed column contain the particle size of 3um, 5um & 10um.
3. Derivatizing column:-
They are installed before an after the analytical column.
Depending upon the position of these columns there are two types.
Pre – Derivatizing column.
Post – Derivatizing column.
Mostly 90% we use post – Derivatizing column.
The main purpose of this column to form derivatives of our sample, so that
our sample/analyte can easily be detected by the detector.
Hence we can say that the main purpose of Derivatizing column is used to
detect/visualize those components that are not detected by ordinary detector.
They contain some reagents which react with sample to give such type of
products which can be easily detected.
e.g. Ninhydrin reagent is used to detect amino acid.
We use florescent tags for the visualization of analyte.
e.g. Dansyl chloride is a florescent material which is extensively used in
florescent tags to detect the sample.
The Derivatizing columns are used in specialized condition, and mostly used
in preparative purpose of HPLC.
4. Micro Column:-
Has diameter less than 1 mm.
These are also used for analytical and small volumes assay.
Advantages:-
Small amount of sample is used.
This is economical.
Normally used in isocratic type of elution.
Disadvantages:-
Not used in gradient elution.
5. Fast Column:-
The basic reasons for using the fast column are given as.
To obtain improved “sample through put” (the amount of sample injected
per unit time). The amount of sample to pass through the column is very
short hence sample through put is high.
To increase the flow rate (it has no effect on the resolution and separation
because of small size and increase sensitivity.
It has same internal diameter as that of analytical column.
It has shorter length and small particle size of 3 um.
Advantages:-
Increased sensitivity (depend on packing of stationary phase).
Increased reproducibility.
Decreased mobile phase usage.
Decreased analysis time.
Independent to flow rate of liquid.
6. Preparative Columns:-
These columns are utilized when the objective is to prepare bulk of sample
for laboratory preparatory applications.
There length is 10 – 30 cm.
Their internal diameter is 10 mm.
The rest composition of the preparative columns is same to the analytical columns.
6. Detectors:-
Detectors are used to convert the column eluent information into electrical
signals but there relationship must be linear. Within the detector the analyte undergoes
some physicochemical interaction by which the analyte is recognized, this interaction is
often optical in nature e.g. being the measurement of the U.V absorbance or
fluorescence of the eluent.
Types of Detectors:
I. Bulk property Detectors(Refractive Index detectors)
II. Partially Specific Detectors(U.V detectors)
III. Totally specific detectors.
Ideal properties of detectors:
1. High sensitivity:
Detector must show high sensitivity (It means that it can detect very minute quantity of
sample in eluent).
2. Selectivity:
Ability of detector to selectively respond to certain compounds and not others.
Ideal detector is selective. They can detect specific compounds because non
specific detector can detect M.P along with sample.
3. Reproducibility:
It means that the result can be obtained by repeating the HPLC process again and again.
e.g. We inject the sample, the sample after passing through whole process the detector
give sharp peaks.
The reproducibility is also known as precision or accuracy.
4. Linear Response & Dynamic Response:
The response or peak increase along with increase conc. of sample. There is
certain range up to which the detector shows linear response and when it goes beyond
this limit, it will not show linear response.
The region where a change in analyte conc. produces a change in output signal
is referred as dynamic response (no relationship between response and conc.).
Fluorescent Phenomenon:-
A relatively small proportion of inorganic and organic compounds exhibit
natural fluorescent where as a large number of pharmaceutical substances and
environmental contaminants show fluorescent property on exposure to electromagnetic
radiations.
Working:-
Light from a fixed wave length U.V lamp passes through a cell, from which
column eluent flows, and act as source.
The light passes into the chamber, through quartz window which is made up of
glass material; it is inert and transparent to U.V rays.
Any fluorescent light emitted is sensed by photoelectric cell positioned normal to
the direction of exciting U.V light.
The photocell senses fluorescent light of all wave length but the wave length of
excitation light can only be changed by use of an alternative lamp.
A more elaborate form of fluorescent detector uses a monochromator to select the
wave length of source light, and second monochromator to select the wave length
of fluorescent light.
Excitation wave length:-
Wave length required by electrons of a compound to reach the higher energy states.
It is specific for every compound to be scanned e.g. excitation wave length of
paracetamol is 257 nm.
Advantages:-
Maximum versatile detector.
Most sensitive detector.
Most specific detector.
Show linear response even at minute quantity.
Applications of HPLC:
HPLC is widely used in many areas of science.
Basically HPLC is employed for
Analytical purpose.
Identification of analyte.
Quantification of analyte.
Resolution of analyte.
Preparative purpose.
Separation of compounds.
Purification of compounds.
1. Pharmaceutical applications:
HPLC is widely used technology in pharmaceutical analysis due to several reasons:
The wide variety of packing materials allows the separation of most
chemical species.
The different types of detectors permit the sensitive detection with
precision and accuracy.
HPLC is employed for the detection of antibiotics, sedatives, steroids
and analgesics. And it is carried for pharmaceutical quality control
testing of drugs, medicines, for compliance, stability studies,
therapeutic drug monitoring etc.
2. Bio chemical applications:-
HPLC is widely used for detection and identification of amino acids,
Proteins, Carbohydrates in urine and blood solution e.g. ELISA test.
3. Food products:-
For the analysis of
Artificial sweeteners like Saccharin.
Antioxidants like Vit.E.
Ensuring soft drink consistency & quality.
Sugar analysis in fruit juices.
Stability of aspartame in the presence of glucose and vanillin.
4. Environmental applications:-
For the detection of :
Phenols in drinking water.
Identification of inorganic and organometallic compounds.
Monitoring of pesticides and herbicides.
Determination of toxicity of tetracycline & tetracycline degradation
products to relevant bacteria.
5. Forensics:-
For the detection of different types of poisons (cyanide, strychnine),
narcotics, alcohol in body fluids/drinks/foods.
Analysis of drug of abuse (heroine, cocaine).
Identification of anabolic steroids in serum, urine, sweat etc,
6. Clinical Purposes:-
Quantification of drugs and their metabolites in biological fluids.
Analysis of antibiotics.
Detection of endogenous neuropeptides in brain, extracellular fluids.
7. Chemical Industry:-
For the analysis of surfactants, propellants and dyes.