High Performance Liquid Chromatography
High Performance Liquid Chromatography (HPLC)
is a form of column chromatography that pumps a sample mixture or analyte in a
solvent (known as the mobile phase) at high pressure through a column with
chromatographic packing material (stationary phase).
It is used in biochemistry and
analytical chemistry to identify, quantify and purify the individual components
of a mixture.
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| Fig: HPLC |
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| Fig: HPLC process |
Stationary phase: The substance on which adsorption of the analyte (the substance to be separated during chromatography) takes place. It can be a solid, a gel, or a solid liquid combination.
Mobile phase: Solvent
which carries the analyte (a liquid or a gas).
Types of HPLC
Based on the phase system (stationary) in the process:
Normal Phase HPLC
This method separates analytes on the basis
of polarity. Stationary phase is polar (hydrophilic) and mobile phase is non-polar
(hydrophobic).
Reverse Phase HPLC
It works on the principle of hydrophobic
interactions hence the more non-polar the material is, the longer it will be
retained. Stationary phase is non-polar (hydrophobic) and mobile phase
is Polar (hydrophilic).
Size-exclusion HPLC
The column is filled with material having
precisely controlled pore sizes, and the particles are separated according to their
molecular size.
Ion-Exchange HPLC
The stationary phase has an ionically charged
surface of opposite charge to the sample ions. This technique is used almost
exclusively with ionic or ionizable samples.
Based on elution technique
1. Isocratic elution
•A separation in which the mobile
phase composition remains constant throughout the procedure is termed isocratic
elution
•In isocratic elution, peak
width increases with retention time linearly with the number of
theoretical plates. This leads to the disadvantage that late-eluting peaks
get very flat and broad.
• Best for simple separations
• Often used in quality
control applications that support and are in close proximity to a
manufacturing process
2. Gradient elution
•A separation in which the mobile
phase composition is changed during the separation process is
described as a gradient elution
•Gradient elution decreases the
retention of the later-eluting components so that they elute faster,
giving narrower peaks. This also improves the peak shape and the peak
height
• Best for the analysis of
complex samples
• Often used in method development
for unknown mixtures
• Linear gradients are most popular
Principle
HPLC is a technique in analytic chemistry
used to separate the components in a mixture, to identify each component and to
quantify each component. It relies on pumps to pass a pressurized liquid
solvent containing the sample mixture through a column filled with a solid
adsorbent material. Each component in the sample interacts slightly different
with the adsorbent material causing different flow rates for the different components
and leading to the separation of the components as they flow out the column.
Instrumentation
1.
SOLVENT RESERVOIR
The
reservoir that holds the mobile phase is called solvent reservoir.
The
typical pore size used in a sinker frit (filter) is on the order of 5 - 10
microns; frits of smaller pore size are too likely to plug. In general, the
mobile phase should be filtered through a 0.3 to 0.5-micron frit after the
solvents and buffers are mixed in order to remove particulate matter.
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| HPLC instrument |
2.
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).
Types
of HPLC Pumps:
a.
Reciprocating
piston pumps:
Consists of a small motor driven piston which moves
rapidly back and forth in a hydraulic chamber that may vary from 35-400μL in
volume.
b.
Syringe
type pump
These are most suitable for small bore columns because
this pump delivers only a finite volume of mobile phase before it has to be
refilled. These pumps have a volume between 250 to 500mL.
c.
Constant
pressure pump
In these types of pumps, the mobile phase is driven through
the column with the use of pressure from the gas cylinder. A low-pressure gas
source is needed to generate high liquid pressures. The valving arrangement
allows the rapid refill of the solvent chamber whose capacity is about 70mL.
3.
INJECTOR
The injector serves to introduce the liquid sample into
the flow stream of the mobile phase for analysis. It is equipped with six port
valves so that a sample can be injected into the flow path at continuous
pressure.
For a manual injector, the knob is manually operated to
deliver the sample to the column. The knob is set to LOAD position for sample
injection using a syringe, the sample is injected into the sample loop, which
is separated from the flow path.
The knob is turned to INJECT position and the eluent
travels through the loop from the pump and delivers the sample to the column.
Typical sample volumes for manual injector are 5-to
20-microliters (μL).
An autosampler is the automatic version for when the user
has many samples to analyze or when manual injection is not practical. It can
continuously inject variable volume of 1 μL-1mL.
4.
COLUMN
Column is considered the “heart of the chromatograph” the
column’s stationary phase separates the sample components of interest using
various physical and chemical parameters.
It is usually made of stainless steel to withstand high
pressure caused by the pump to move the mobile phase through the column packing
other material include PEEK and glass
The small particles inside the column are called the
“packing” what cause the high back pressure at normal flow rates.
Column packing is usually silica gel because of its
particle shape, surface properties, and pore structure give us a good
separation.
Other material used include alumina, a
polystyrene-divinyl benzene synthetic or an ion-exchange resin
Dimensions of the analytical column are usually
-straight, Length (5 to 25 cm), diameter of column (3 to 5 mm), diameter of
particle (35μm). Number (40 k to 70 k plates/m)
Guard
column is used to remove
particular matter and contamination, it protect the analytical column and
contains similar packing its temperature is controlled at < 150 °C, 0.1 °C
5.
DETECTOR
Ultraviolet (UV)
This type of detector responds to substances that absorb
light. UV detector is mainly to separate and identify the principal active
components of a mixture. These are the most versatile, having the best
sensitivity and linearity. UV detectors cannot be used for testing substances
that are low in chromophores (colorless or virtually colorless) as they cannot
absorb light at low range. They are cost-effective and popular and are widely
used in industry.
Fluorescence
This is a specific detector that senses only those
substances that emit light. This detector is popular for trace analysis in environmental
science.
As it is very sensitive, its response is only linear over
a relatively limited concentration range. As there are not many elements that
fluoresce, samples must be syntesized to make them detectable.
Mass Spectrometry
The mass spectrometry detector coupled with HPLC is
called HPLCMS. HPLC-MS is the most powerful detector, widely used in
pharmaceutical laboratories and research and development.
The principal benefit of HPLC-MS is that it is capable of
analyzing and providing molecular identity of a wide range of components.
Refractive Index (RI) Detection
The refractive index (RI) detector uses a monochromator
and is one of the least sensitive liquid chromatography detectors. This
detector is extremely useful for detecting those compounds that are non-ionic,
do not absorb ultraviolet light and do not fluoresce e.g. sugar, alcohol, fatty
acid and polymers.
6.
DATA COLLECTION DEVICE
Computer collects all the data from the
detectors and gives the corresponding results.
Parameters
·
THEORETICAL
Theoretical parameters are
largely derived from two sets of chromatographic theory: plate theory (as part
of Partition
chromatography), and the rate theory of chromatography
/ Van
Deemter equation.
Retention
time (tR )
Retention time is a measure of the time taken for a solute to pass
through a chromatography column. It is calculated as the time from injection to
detection. This is the sum of the total times the components spends in
the mobile phase (t0) and in the
stationary phase.
Retention
(Capacity) Factor (k)
The retention (or
capacity) factor (k) is a means of measuring the retention of an analyte on the
chromatographic column.
It is the ratio of the
time the component spends in the stationary phase to time in the mobile phase.
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| Determination of Retention factor |
A high k
value indicates that the sample is highly retained and has spent a significant
amount of time interacting with the stationary phase.
Chromatographers
like to keep k values between 1 and 10 for good separations.
Retention volume:
Retention volume is the volume of
carrier gas required to elute 50% of the component from the column. It is the
product of retention time and flow rate.
Retention volume =
Retention time × flow rate
Resolution (Rs)
Resolution is the measure of
extent of separation of 2 components and the base line separation achieved. A
resolution value of 1.5 or greater between two peaks will ensure that the
sample components are well (baseline) separated to a degree at which the area
or height of each peak may be accurately measured.
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| Determination of Resolution |
Selectivity
(Separation) Factor (α)
The
selectivity (or separation) factor (α) is the ability of the chromatographic
system to ‘chemically’ distinguish between sample components. It is usually
measured as a ratio of the retention (capacity) factors (k) of the neighboring two
peaks
.
 |
| Determination of Separation factor |
The selectivity Factor (α) is always greater than one
– as when α is equal to one, the two peaks are co-eluting (i.e. their retention
factor values are identical). The greater the selectivity value, the further
apart the apices of the two peaks become.
Efficiency
Efficiency factor practically measures how sharp component
peaks on the chromatogram are. Efficiency factor is synonymous with
plate number, and the 'number of theoretical plates'.
Efficiency of a column is
expressed by the theoretical plates (N).
 |
 |
| Determination of theoretical plates |
If number of theoretical plates is
high, the column is said to be highly efficient. In HPLC, high values like
40,000 to 70,000/meter are recommended.
Height
Equivalent to a Theoretical Plate (HETP):
A theoretical
plate is an imaginary or hypothetical unit of a column where distribution of
solute between stationary phase and mobile phase has attained equilibrium. It
can also be called as a functional unit of the column. A theoretical plate can
be of any height, which describes the efficiency of separation. If HETP is
less, the column is more efficient. If HETP is more, the column is less
efficient.
HETP
= length of the column/ no. of theoretical plates
Asymmetry factor:
A
chromatographic peak should be symmetrical about its centre and said to follow
Gaussian distribution. But in practice due to some factors, the peak is not
symmetrical and shows tailing or fronting.
Tailing is due to more active adsorption sites and can be eliminated by support pretreatment.
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| Determination of Asymmetry factor |
Asymmetry factor
(0.95 to 1.05) can be calculated by
As = B/A (B, A calculated
by 5% or 10% of the peak height).
Broad peaks
occur due to the more conc. Of sample, large injection volume, column
deterioration.
Ghost peaks
occur due to the contamination of the column, compound from earlier injections.
Negative peaks
occur if mobile phase absorbance is larger than sample absorbance.
Peak doubling
occurs due to the co- elution of interfering compound, column over load, channeling
in column.
Base line spikes
occur due to the air bubbles in the mobile phase and/or detector, column
deterioration.
·
INTERNAL DIAMETER
The internal diameter (ID)
of an HPLC column is an important parameter that influences the detection
sensitivity and separation selectivity in gradient elution. It also determines
the quantity of analyte that can be loaded onto the column.
·
PARTICLE SIZE
Most traditional HPLC is performed with the stationary phase attached to
the outside of small spherical silica particles (very small beads).
These particles come in a variety of sizes with 5 µm beads being the most
common. Smaller particles generally provide more surface area and better
separations.
·
PORE SIZE
Many stationary phases are porous to provide greater surface area. Small
pores provide greater surface area while larger pore size has better kinetics,
especially for larger analytes. For example, a protein which is only slightly
smaller than a pore might enter the pore but does not easily leave once inside.
·
PUMP PRESSURE
Pumps vary in pressure capacity, but their performance is measured on
their ability to yield a consistent and reproducible volumetric
flow rate. Pressure may reach as high as 60 MPa
(6000 lbf/in2), or about 600 atmospheres.
Modern HPLC systems have been improved to work at much higher pressures, and
therefore are able to use much smaller particle sizes in the columns
(<2 μm). These ultra-high performance liquid chromatography"
systems or UHPLCs can work at up to 120 MPa (17,405 lbf/in2),
or about 1200 atmospheres.
·
DETECTORS
HPLC detectors fall into two main categories: universal or selective. Universal
detectors typically measure a bulk property (e.g. refractive index) by measuring a difference of a physical property between the mobile phase and
mobile phase with solute while selective detectors measure a solute property
(e.g. UV-Vis absorbance) by simply responding to the physical or chemical property of the solute.
·
Autosamplers
Large numbers of samples can be automatically injected onto an HPLC
system, by the use of HPLC autosamplers. In addition, HPLC autosamplers have an
injection volume and technique which is exactly the same for each injection,
consequently they provide a high degree of injection volume precision.
Application
Pharmaceutical
applications
1.
To control drug stability.
2.
Tablet dissolution study of
pharmaceutical dosages form.
3.
Pharmaceutical quality
control.
4.
Shelf life determinations
of pharmaceutical products.
5.
Identification of
counterfeit drug products.
Environmental
applications
1.
Phenols in Drinking Water.
2.
Identification of diphenhydramine in sediment samples.
3.
Biomonitering of PAH pollution in high-altitude mountain
lakes through the analysis of fish bile.
4.
Estrogens in coastal waters - The sewage source.
5.
Toxicity of tetracyclines and tetracycline degradation
products to environmentally relevant bacteria.
6.
Assessment of TNT toxicity in sediment.
Forensic applications
1.
A mobile HPLC apparatus at dance parties - on-site
identification and quantification of the drug Ecstasy.
2.
Identification of anabolic steroids in serum, urine,
sweat and hair.
3.
Forensic analysis of textile dyes.
4.
Determination of cocaine and metabolites in meconium.
5.
Simultaneous quantification of psychotherapeutic drugs in
human plasma.
Clinical applications
1.
Quantification
of DEET in Human Urine.
2.
Analysis
of antibiotics.
3.
Increased
urinary excretion of aquaporin 2 in patients with liver cirrhosis.
4. Detection of endogenous neuropeptides in
brain extracellular fluids.
Food and Flavor
1.
Ensuring soft drink consistency and quality.
2.
Analysis of vicinal diketones in beer.
3.
Sugar analysis in fruit juices.
4.
Polycyclic aromatic hydrocarbons in Brazilian vegetables
and fruits.
5.
Trace analysis of military high explosives in
agricultural crops.
6. Stability of
aspartame in the presence of glucose and vanillin.
Advantages of HPLC
1.
Separations
fast and efficient (high resolution power)
2.
Continuous
monitoring of the column effluent
3.
It can
be applied to the separation and analysis of very complex mixtures
4.
Accurate
quantitative measurements.
5.
Repetitive
and reproducible analysis using the same column.
6.
Adsorption,
partition, ion exchange and exclusion column separations are excellently made.
7.
HPLC is
more versatile than GLC in some respects, because it has the advantage of not
being restricted to volatile and thermally stable solute and the choice of
mobile and stationary phases is much wider in HPLC.
8.
Both
aqueous and non-aqueous samples can be analyzed with little or no sample
pretreatment.
9.
A
variety of solvents and column packing are available, providing a high degree
of selectivity for specific analyses.
10. It provides a means for determination of
multiple components in a single analysis.
