HPLC Chromatography

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HPLCChromatography
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Chromatography

• A diverse and important group of methods that
  permit separation of closely related components
  in complex mixtures
• A powerful separation tools
• Qualitative (preparative, purification)
• Quantitative

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Chromatography

• Sample is dissolved in a mobile phase (gas,
  liquid or supercritical fluid).
• This mobile phase is then forced through an
  immiscible stationary phase, which is fixed in
  place in a column or a solid surface.

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Classification

• Two ways
• Physical means by which the stationary and mobile
  phase are bought into contact (e.g.. column vs.
  planar chromatography)
• A more fundamental mean based on the types of
  mobile and stationary phases and the kinds of
  equilibria involved in the transfer of solutes
  between phasesHO1.

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Three General Categories

• Liquid Chromatography (plane surfaces and
  column)
• Gas Chromatography (only in column)
• Supercritical-fluid Chromatography (only in
  column)

Today we will cover the liquid chromatography
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Liquid Chromatography

• Plane surfaces (e.g. paper, TLC)
• Column chromatography (hand-packed or pre-packed
  columns)

• The solvent moves through
• the paper/thin gel, drawn by
• capillary action

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Elution on Column Chromatography
Elution involves transporting a species through a
column by continuous addition of fresh mobile
phase.
Elution can be achieved by gravity force
(gravity flow, and it can take a long time to
complete
A chromatogram
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Elution on Column Chromatography
Original column LC was with glass column with
gravity flow the technique Is still good for
preparative and purification purposes.
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Applications of Liquid-Chromatography
Skoog/Leary, 1992
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Applications of Liquid-Chromatography

• Exclusion chromatography (MW gt10,000)
• Ion-exchange (Low molecular weight ionic species)
• Adsorption (non-polar species)
• Partition methods (small polar but nonionic
  species)

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Liquid-Column Chromatography

• LC can be carried out using a glass tube
  hand-packed with a stationary phase (solid)
  through which a solvent is allowed to
  gravity-flow.
• So why do we need all the complicated high-tech
  equipment?
• One Word SPEED
• A single analysis by "Classical" LC can take
  anywhere from 2 to 12 hours to carry out.
• HPLC allows an equivalent analysis to be done in
  2 to 12 minutes.
• Reproducibility. A classical column must be
  freshly packed for each analysis, increasing the
  chance of errors. A single HPLC column can be
  used for hundreds or thousands of samples.

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What is HPLC?

• High-Performance Liquid Chromatography
• Developed in 1960s as faster way to do column
  chromatography
• Advantages over traditional chromatography
  include
• Speed
• Adaptability
• resolution
• sensitivity
• columns reusable

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HPLC

• Popularity
• Widely applicable to numerous fields of study
  both academic, industrial, and biomedical.
• Great for separation of non-volatiles
• Amino acids, proteins, nucleic acids,
  hydrocarbons, carbohydrates, pharmaceuticals,
  pesticides, terpenids, pigments, antibiotics,
  steroids, vitamins, and various other organic and
  inorganic substances.
• Generally, if a compound can be solublized in
  common solvents such as water, alcohol,
  acetonitrile, acetone then HPLC can probably be
  used.

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HPLC

• One of the most widely used analytical separation
  techniques.
• Uses a liquid mobile phase to separate components
  in a mixture
• Used high or low pressure to push solvent through
  a separation column
• Popular because
• Sensitive
• Accurate, quantitative methods can be used
• Great for separation of non-volatile components,
  heat labile compounds, and semi-volatile
  compounds.
• Non-destructive

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Basic Hardware Components of HPLC

• Solvent Delivery System (Pump)
• Injector (introduce samples)
• Column guard
• Column (containing stationary phase)
• Detectors (eyes )
• Waste Collector
• Recorder (Data Collection)

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Diagram of HPLC
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Old Time HPLC!
Integrator (recorder)
Line filter
Detectors
Solvent reservoir
Pump
Injector
Column
Column guard
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Modern HPLC
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Pumps

• Ideal pumps
• Ability to generate high pressure
• Pulse-free output
• Accurate control of flow
• Corrosion resistant

• Role Deliver the mobile phase
• Two groups of pumps
• Constant pressure
• Constant volume
• Three types of pumps are available
• Reciprocating pumps (90 of Commercial HPLC
  produce pulse flow)
• Displacement pumps (produce flow that are
  independent of viscosity and
• back pressure)
• 3) Pneumatic pumps (cannot do gradient and
  pressure less than 2000psi)

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Injectors
Most common injector is sample loop (5-500uL
0.5-5uL)
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Columns

• Analytical column variables
• Length (10-30 cm)
• ID (4-10mm)
• Packing (many kinds)
• Particles sizes (3-10 µm)pore sizes
• Most common columns 25cm x 4.6 id with 5µm
  particles
• Preparative columns

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Detectors

• Visualize separated compounds and translate the
  concentration changes into signals
• Using every conceivable physical and chemical
  properties
• Characteristics of an ideal detector
• Adequate sensitivity
• Good stability and reproducibility
• Gives linear response to analysts that have
  several ranges magnitudes
• Short response time
• High reliability and ease of use
• Similarity in response toward all analyst
• Non-destructive

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Detectors

• Two basic types of detectors
• Bulk property detectors response to a
  mobile-phase bulk property
• Refractive index
• Dielectric constant
• Density
• Solute property detectors response to solute
  property
• Spectroscopy (IR, UV, MS, Fluorescence)

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Most Common HPLC Detectors

• Absorbance (Absorption of UV-Vis based on Beers
  law)
• Fluorescence
• Electrochemical
• Refractive index
• Conductivity
• Mass spectrometry
• FI-IR
• Light scattering
• Others (under development, not on the market)

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Popular Types of Detectors (Top 4)

• UV/Visible absorption detectors
• Fluorescence
• Refractive Index (RI)
• Electrochemical (ED)

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UV/Vis Absorbance Detectors

• Compounds with strong UV/Vis chromophores
• Compounds with conjugated or nonconjugated double
  bonds aromatic molecules
• AD Simple, reliable, inexpensive, compatible
  with gradient elution and non-destructive
• Dis-AD Not as sensitive as fluorescence
  detection, ED, not-universal (only for molecules
  with chromophores)
• Samples of use vitamins, carotenoids,
  phytonutrients

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UV/Vis Absorbance Detectors

• uv/vis - you can purchase
• fixed wavelength
• variable wavelength
• diode array

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UV/Vis Absorbance Detectors
Light sources Deuterium or tungsten filament
sources
The Variable Wavelength UV Detector uses a
monochromator (slits and a grating) to select one
wavelength of light to pass through the sample
cell.
The Photodiode Array Detector passes all
wavelengths of light through the sample cell,
then focuses each wavelength on a single sensor
element.
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Absorbance Detector Output
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Fluorescence Detectors

• Compounds with fluorophors
• By nature (carbamate pesticides, aflatoxins,
  vitamins, amino acids)
• By post-column derivatization
• AD Highly sensitive (femtomole 10-15), low
  background, highly selective (two distinct wave
  lengths instead of one in Ab detector), can solve
  co-elution problems, post-column derivatization
  can be used for this detection
• Dis-AD Perceived difficulty of its use, more
  instrumental variables to account for during
  optimization, changes in fluorescence can occur
  with pH and viscosity
• Samples of use vitamins E, drugs, aflatoxins

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Fluorescence Detector Configuration
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Refractive Index Detectors

• Compounds that do not have strong UV/Vis
  chromophores, fluorophours, electrochemical
  activity or ionic conductivity
• AD Universal in nature
• Dis-AD Lack of sensitivity impractical for
  gradient elution instability of base line
• Samples of use organic acids, sugars, fungal
  metabolites, oligosaccharides

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Recorder/Data Collections

• Many recoding devices are available
• Strip-chart recorder (retention time/Peak areas
  or peak height)
• Integrator
• Computer controlled data collections