High Performance Liquid Chromato: School of Basic and Sciences

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SCHOOL OF BASIC AND SCIENCES

Department of chemistry
High Performance Liquid
Chromatography
Presentation by
Maruthanayagam. R
Register No. D190504
PG Diploma in Chemical Lab Technician
Central University of Tamil Nadu
Evolution of HPLC
• 1903 - The Russian botanist Mikhail Tsvet is considered to have
‘invented’ the chromatographic technique when he reported
separations of different plant pigments into a series of colored bands
on a packed column. He called this technique ‘chromatography’.
• The origins of HPLC date back to the invention of chromatography in
the early 20th century, through the introduction of partition and
paper chromatography in the 1940s, to the introduction of liquid
chromatography in the early 1960s. Shortly thereafter, the need for
better resolution and high-speed analyses of nonvolatile samples led
to the development of HPLC.
• 1969 - The first commercial HPLC was manufactured by
Waters Corporation, and was known as the ALC100 HPLC.
Jack Kirkland (left), with technician Glen Wallace, in front of a homebuilt HPLC instrument in 1967.
Introduction
• HPLC stands for“High-performance liquid chromatography”(sometimes referred to
as High- pressure liquid chromatography).

• High performance liquid chromatography is a powerful tool in analysis, it yields


high performance and high speed compared to traditional columns
chromatography because of the forcibly pumped mobile phase.

• HPLC is a chromatographic technique that can separate a mixture of compounds

• It is used in biochemistry and analytical chemistry to identify, quantify and purify


the individual components of a mixture.
• 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.
PRINCILPE OF HPLC
•To understand the principle of HPLC, we must first
look at the principle behind liquid chromatography
•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 main principle of separation is adsorption.
•When a mixture of components are introduced into the column . various chemical
and/or physical interactions take place between the sample molecules and the particles
of the column packing .
•They travel according to their relative affinities towards the stationary phase.
•The component which has more affinity towards the adsorbent, travels slower.
•The which has less affinity towards the stationary phase travels faster.
•Since no two components have the same affinity towards the stationary phase, the
components are separated
HPLC System

Flow chart of HPLC mechanism


Instrumentation of HPLC

Solvent
reservoirs
1 and degassing

2
5
3
4

1 – Pump
2 – Injector
3 – Column
4 – Detector
5 – Computer
Picture of HPLC instrument
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).
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


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 .
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:
3. User loads vials filled with sample solution into the auto sampler tray
(100 samples)
4. 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 for unattended
automatic operation
Manual Injectors
Sample Loop

Load - Inject

Front View Rear View

Inject
Automatic Injectors

Step 1 Step 2

Step 3
HPLC 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.
• Within the Column is where separation occurs.
• 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.
Column types
• Normal phase

• Reverse phase

• Size exclusion

• Ion exchange
Normal phase STATIONARY PHASES
(NORMAL POLARITY)

• In this column type, the Silica or alumina possess polar sites that
retention is governed by interact with polar molecules.
the interaction of the silica
polar parts of the O
stationary phase and Polar Group HO Si

solute. O

• For retention to occur in


normal phase, the Components elute in increasing
order of polarity.
packing must be more
polar than the mobile
phase with respect to the Most polar…….Least polar
sample.
Reverse phase STATIONARY PHASES
(REVERSE POLARITY)
• In this column the packing material is
relatively nonpolar and the solvent is polar If the polar sites on silica or alumina are
with respect to the sample. Retention is the capped with non-polar groups, they interact
result of the interaction of the nonpolar
components of the solutes and the nonpolar strongly with non-polar molecules.
stationary phase.
• Typical stationary phases are nonpolar Silica
hydrocarbons, waxy liquids, or bonded C18 phase Me O
hydrocarbons (such as C18, C8, etc.) and the Si O Si
solvents are polar aqueous- organic mixtures Me O
such as methanol-water or acetonitrile-water.
Common Reverse Phase Solvents
Components elute in decreasing
Methanol CH3OH order of polarity.
Acetonitrile CH3CN

Tetrahydrofuran Most non-polar…….Least non-polar


Water H2O
Sizeexclusion STATIONARY PHASES
(SIZE EXCLUSION)
• In size exclusion the HPLC Size exclusion gels separate on the basis of molecular
column is consisted of substances size. Individual gel beads have pores of set size, that
which have controlled pore sizes restrict entry to molecules of a minium size.
and is able to be filtered in an
ordinarily phase according to its
molecular size.

• Small molecules penetrate into the


pores within the packing while
Large molecules elute fast (restricted path),
larger molecules only partially while small molecules elute slowly (long path
penetrate the pores. The large length)
molecules elute before the Larger molecules…….Smaller molecules
smaller molecules.
Ion exchange
• In this column type the sample components are separated based
upon attractive ionic forces between molecules carrying charged
groups of opposite charge to those charges on the stationary phase.

• Separations are made between a polar mobile liquid, usually water


containing salts or small amounts of alcohols, and a stationary phase
containing either acidic or basic fixed sites.
IONIC EXCHANGE
Modes of High Performance Liquid
Chromatography
Types of Compounds Mode Stationary Phase Mobile Phase
Neutrals Reversed Phase C18, C8, C4 Water/Organic
Weak Acids cyano, amino Modifiers
Weak Bases
Ionics, Bases, Acids Ion Pair C-18, C-8 Water/Organic Ion-Pair
Reagent
Compounds not soluble Normal Phase Silica, Amino, Cyano, Organics
in water Diol
Ionics Inorganic Ions Ion Exchange Anion or Cation Aqueous/Buffer
Exchange Resin Counter Ion
High Molecular Weight Size Exclusion Polystyrene Silica Gel Filtration-
Compounds Aqueous
Polymers Gel Permeation-
Organic
Detector:
• The detector can detect the individual molecules that elute from the
column and convert the data into an electrical signal
• A detector serves to measure the amount of those molecules
• The detector provides an output to a recorder or computer that results
in the liquid chromatogram
• Detector is selected based on the analyte or the sample under
detection
Commonly used detectors in HPLC
Ultraviolet (UV)
• This type of detector responds to substances that absorb light.
• The UV detector is mainly to separate and identify the principal active
components of a mixture.
• UV detectors 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.
Data processing unit (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 determine the time of elution (retention time)
of the sample components (qualitative analysis) and the amount of
sample (quantitative analysis).
• The concentration of each detected component is calculated from the
area or height of the corresponding peak and reported.
Applications
HPLC is one of the most widely applied analytical separation
techniques.

Pharmaceutical:
• Tablet dissolution of pharmaceutical dosages.
• Shelf life determinations of pharmaceutical products.
• Identification of counterfeit drug products.
• pharmaceutical quality control.
Environmental:
• Phenols in Drinking Water.

• Identification of diphenhydramine in sediment samples.

• Biomonitering of PAH pollution in high-altitude mountain lakes through the analysis of fish
bile.

• Estrogens in coastal waters - The sewage source.

• Toxicity of tetracyclines and tetracycline degradation products to environmentally relevant


bacteria.

• Assessment of TNT toxicity in sediment


Forensics:
•A mobile HPLC apparatus at dance parties - on-site identification and
quantification of the drug Ecstasy.
•Identification of anabolic steroids in serum, urine, sweat and hair.
•Forensic analysis of textile dyes.
•Determination of cocaine and metabolites in meconium.
•Simultaneous quantification of psychotherapeutic drugs in human
plasma.
Clinical:
•Quantification of DEET in Human Urine.
•Analysis of antibiotics.
•Increased urinary excretion of aquaporin 2 in patients with liver cirrhosis.
•Detection of endogenous neuropeptides in brain extracellular fluids.

Food and Flavor:


• Ensuring soft drink consistency and quality.
• Analysis of vicinal diketones in beer.
• Sugar analysis in fruit juices.
• Polycyclic aromatic hydrocarbons in Brazilian vegetables and fruits.
• Trace analysis of military high explosives in agricultural crops.
• 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 pre treatment
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.
References
• Hearn M.T.W. Ion-pair chromatography on normal and reversed-phase systems. Adv.
Chromatogr. 1980; 18: 59–100.
• Bergh J. J., Breytenbach, J. C. Stability-indicating High-performance Liquid- chromatographic
Analysis of Trimethoprim in Pharmaceuticals. J. Chromatogr. 1987; 387: 528-531.
• Rodenas V., Garcia M.S., SanchezPedreno C., Albero M.I. Flowinjection spectrophotometric
determination of frusemide or sulphathiazole in pharmaceuticals. J. Pharm. Biomed. Anal.
1997; 15: 16871693.
• Shah A.J., Adlard M.W., Stride J.D. A sensitive assay for clavulanic acid and sulbactam in
biological fluids by highperformance liquid chromatography and precolumn derivatization. J.
Pharm. Biomed. Anal. 1990; 5: 437443.
• https://cen.acs.org/articles/94/i24/50-years-HPLC.html
• Ayerton J. Assay of ceftazidime in biological fluids using high-pressure liquid chromatography.
J. Antimicrob. Chemother. 1981; 8: 227-231.
THANK
YOU

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