HPLC

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3.

High Performance of Liquid Chromatography (HPLC)

Classical Liquid Chromatography

 SP is held in a narrow tube

 The solvent passes though SP


(carrying the sample) by the
action of gravity under
atmospheric pressure

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Limitation of classical LC

 The columns are used once


 makes the column packing tedious and expensive

 Packing materials have large particle size (150-250 m)


 low column efficiency

 MP flow rate cannot be adjusted (gravity action)

 Lengthy operation time and manual analysis solutes


 Labor intensive, time consuming

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High performance liquid chromatography (HPLC)

 Developed in the mid 1970’s


 LC with increased efficiency
 Uses small size (3-10 μm) stationary material uniformly
packed in a column of very small diameters (3-5 mm)

 A high pressure is applied to force the MP through the


tightly-packed column
Samples moves smoothly (non-pulsed)

 Columns are reusable


 Flow rate adjustment is possible

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Instrumentation of HPLC

Fig.1. Example of HPLC instrument with insets showing


various parts
High Performance Liquid Chromatography (HPLC)

Fig.2. Schematic block diagram showing each parts of


HPLC
1. Reservoirs

 Reservoir is a glass (or stainless steel) bottle which contains


the MP (solvent)
 Mobile phase should be:
 Inert towards the sample components and the SP
 Less viscous (reduces pressure)
 Highly pure (free from impurities and dissolved gases
 Impurities: causes interference in the separation and
detection
 Dissolved gases: form bubbles which interfere in the
pumping of the MP and in the detection of analytes
 Removal of dissolved gases = degassing
 are removed from the solution by bubbling of an
inert gas of low solubility like Helium
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MP composition: Isocratic versus gradient elution

 Gradientelution: continuous
change of solvent composition to
increase MP strength

 Isocratic elution: performed


with a single solvent (or
constant solvent mixture)

Fig. 3. Improvement in separation


effectiveness of Gradient elution7
2. Pump
 The quality of a pump is measured by
how steady and reproducible a flow it
can produce. A fluctuating flow
creates detector noise that obscures
weak signals.
 Requirements of pumping system:
 Create non-pulsing solvent flow.
 Flow rates from 0.1-10 mL/min.
 Flow control and flow
reproducibility of a maximum Reciprocating pump (in
error of 0.5%. 90% of systems)
 Corrosion resistant (stainless steel
and Teflon).
 Generation of high pressure up to
6000 psi (400 Bar) 8
3. Injector
When the valve is rotated 60° counterclockwise, the content of the
sample loop is injected into the column at high pressure.

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Sample preparation

 In general, the “dilute and shoot” approach can be used


for most liquid samples

 A common process of “grind → extract → dilute → filter”


is used for most solid sample

 More complex samples, such as lotions, creams, and


physiological samples (serum or plasma) might require
additional sample clean-up and extraction such as liquid-
liquid extraction or solid-phase extraction (SPE).

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4. The Column
 It is made of stainless-steel tubing
 Column length is: 10 – 30 cm and internal
diameter is: 4 – 10 mm
 Column packing typically have particle
sizes 5 or 10 µm. Column of this type often
contain 40000-60000 plates/m
 It is expensive and easily damaged by
dust or particles in the sample or solvent.
Protection:
1. Periodically renew guard columns
containing the same SP like main
column.
2. Pass solvents and samples through filters 11
van Deemter for HPLC

H ∞ A + C

Eddy diffusion (A-term) Mass transfer (C-term)


5. Detection
 Requirements of a detector:
 Sensitive to low concentrations of every analyte.
 Small volume to avoid peak broadening.
 Linear response.
 Insensitive to changes in temperature or solvent
composition.

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5.1 UV detector

 Use ulteraviolet radiation source in a


flow cell
 Employ deuterium, tungsten or
xenon lamps with monochromators to
choose the optimum λ.

The flow cell

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5.2. Fluorescence detector

 Fluorescence is measured after


excitation of the eluate with a
laser.
 Advantage: very sensitive
 Disadvantage:
response to few analytes.
Derivatization of originally non-
fluorescing analytes by covalent
attachment of fluorophores to
analytes either prior to
chromatographic separation, or by
adding derivatization reagents to
the eluate between column and
detector.

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Partition Chromatography
 The separation can be performed into two ways based on
stationary phase (normal phase or reverse phase)
 In normal-phase chromatography, the least polar component is
eluted first; increasing the polarity of the mobile phase then
decreases the elution time. In contrast, with reversed-phase
chromatography, the most polar component elutes first, and
increasing the mobile phase polarity increases the elution time.

Bonded-phase packing
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Problem
Consider the following three compounds
HOCH2CH2OH CH3CH2CH2CH2CH2CH3 C6H5CH3
(ethylene glycol) (hexane) (toluene)
where toluene has a polarity that is in between the two other
compounds.

i) In what order would the compounds elute in normal-phase


HPLC? For a given mobile phase composition, the retention
time of ethylene glycol is 5.6 minutes. Will the retention time
increase or decrease by increasing the polarity of the mobile
phase?

ii) In what order would the compounds elute in reversed-phase


HPLC? For a given mobile phase composition, the retention
time of hexane is 5.6 minutes. Will the retention time increase
or decrease by increasing the polarity of the mobile phase?
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Adsorption Chromatography

Stationary phase (adsorbents):


 The most common are alumina and silica gel in which the
interactions with solute molecules is due to OH groups present
on their surface.

 More polar molecules are adsorbed more strongly and will


elute more slowly

 Strength of adsorption of polar groups (solutes) on polar


support is in the following order:

Olefins<Ethers<Esters<Lactones<Aldehydes<Amines<Phenols<Acids.

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Applications in separation of natural products

 Alumina: sterols, dyestuffs, vitamins, esters, alkaloids


and inorganic compounds.
 Not used for compounds containing phenolic or
carboxylic groups

 Silica gel: sterols and amino acids

 Carbon: peptides, carbohydrates and amino acids

 Calcium carbonate: carotenoids and xanthophylls

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Selection of the solid support

The support material should:

 adsorb and retain the stationary phase.


 expose as large surface as possible to the MP
 be mechanically stable.
 be easy to pack.
 not retard the solvent flow

Examples of solid supports:


Silica gel, diatomaceous earth and cellulose

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Size-exclusion or gel permeation chromatography
 Porous polymeric matrix: formed of spongy particles,
with pores completely filled with the liquid mobile
phase (gel).

 The gels (polymers) consist of open, three-dimensional


networks formed by cross-linking of long polymeric
chains.

 The pore size varies with the degree of cross-linking.

 The diameter of the pores is critical as separation is


based on that molecules above certain size are totally
excluded from the pores because they can not enter
the gel.

 The interior of the pores is reached, partially or


wholly, by smaller molecules. 21
Mobile phase

Mobile phase is a liquid as water or dilute alcohol

Separation mechanism

 Based on difference between the solute molecular


weights.

 Molecules will distribute themselves outside and inside the


pores according to their size.

 Larger are excluded, medium sized enter half-way and


smallest permeate all the way.

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Applications of GPC to natural products

 Separation of mixture of mono- and polysaccharides.

 Separation of amino acids from peptides and proteins.

 Separation of proteins of different molecular weights.

 Separation of polysaccharides and soluble RNA.

 Separation of myoglobin and haemoglobin.

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Ion Exchange Chromatography
 Process by which ions of an electrolyte solution are brought
into contact with an ion exchange resin.

 The ion exchange resin is an insoluble polymer consisting of


a "matrix“ that carries
 fixed charges (not exchangeable)
 mobile active ions "counter ions" which are loosely
attached to the matrix.

 In water, the counter-ions move more or less freely in the


framework and can be replaced by ions of the same sign
present in the surrounding solution.

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Cation Exchangers
 Active ions (counter ions) are cations.
The polar groups attached to the matrix are acidic
(sulphonic acids, carboxylic acids, phenols, phosphoric acids)

Example: a cation exchanger in the free carboxylic


acid form:

Example: X-COO- H+
 X = Frame work (matrix)
 -COO- = fixed charge (anionic), non-exchangeable
 H+ = counter ion (cation), Exchangeable

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Regeneration of the resin
 Ion exchange process is generally reversible e.g in the
following:

X-2Na+ + Ca2+ 2Cl - → X-Ca2+ + 2Na+ Cl –

 The cation exchanger could be exhausted after exchanging


all Na+ for Ca2+

 the exchanger could be regenerated (loaded again with Na+)


by contacting it with excess Na+ ions e.g. a solution of NaCl.

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Anion Exchangers
 Active ions (counter ions) are anions.
 The polar groups attached to the matrix are tertiary or
quaternary ammonium groups (basic).

 Example: Anion exchanger in the quaternary ammonium


form:
Example: X-NR3+OH –

 X = Framework (matrix)
 -NR3+ = Fixed charge (cationic), non-exchangeable
 -OH– = counter ion (anion), Exchangeable

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Applications of Ion Exchange Chromatography

 Water softening:Removal of Ca2+, Mg2+ and other


multivalent ions causing hardness of water by filtration
through a layer of strong cation resin.

 Water demineralization: Removal of cations and


anions dissolved in water. Usually carried by the two step
technique in which two columns of strongly acid cation
exchanger in [H+] form and strongly basic anion exchanger
in [OH-] form are used in sequence.

 Neutralization: Cationic exchanger in [H+] can be used to


neutralize alkali hydroxide and anionic exchanger in [OH-]
form to neutralize the acidity.

 Separation of electrolytes from non-electrolytes


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Affinity Chromatography

 Analytes are separated based on


biochemical interactions (enzyme-substrate
interaction)

 The SP has a biochemical that has affinity


to a specific type of analyte

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