Liquid Chromatography HPLCUPLC

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Liquid ChromatographyHPLC/UPLC

• Kevin Lankford
• Chem 6200
• Topics in Analytical

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Liquid Chromatography

• There are many ways to classify Liquid
  Chromatography (LC). Normally it is described by
  the nature of the stationary phase and separation
  process.
• Ion exchange chromatography
• Size exclusion chromatography
• Adsorption chromatography

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Ion exchange

• Stationary bed has particles with a charged
  surface opposite of sample ions.
• Exclusively used for ionizable samples
• Buffers are used as the mobile phase using pH and
  ionic strength to control the elution time.

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Size exclusion chromatography

• These columns are filled with a particular pore
  size serving to filter the sample
• Large molecules wash through the column faster
  than the smaller ones giving the larger molecules
  a lower retention time.

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Adsorption Chromatography

• These columns are packed with a stationary phase
  such as silica that serves as an adsorbent to the
  specific compound.
• This separation is based on adsorption and
  desorption steps using different solvent ratios
  to interact with the sample.

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Different Phases

• Normal Phase This is where the stationary bed
  is strongly polar (silica gel) and the mobile
  phase is largely non-polar such as hexane or THF.
• Reverse Phase The stationary phase is non-polar
  and the mobile phase are polar liquids such as
  methanol, acetonitrile, or water. The more
  non-polar substances have longer retention.

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Elution Types

• Isocratic where the eluent is at a fixed
  concentration.
• Gradient where the eluent concentration and
  strength are changing.

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Types of Liquid Chromatography


Gravity Chrom. Tsvett, 1903
Flash Chrom. 1978
HPLC 1952
UPLC 2004
(TLC) Paper Chrom.
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HPLC Characteristics

• Columns have small internal diameters (2-10 mm)
  usually made with a reusable material like
  stainless steel
• High inlet pressures of several thousand psis
  and controlled flow of mobile phase
• Precise sample introduction and small sample
  requirements
• Special continual flow detectors that use small
  flow rates and low detection limits
• Some are equipped with automated sampling devices
• Rapid analysis with high resolution

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Stationary Phase in HPLC

• Particle size 3 to 10 µm packed tightly with a
  pore size of 70 to 300 Å
• Surface area of 50 to 250 m2/g
• Bond phase density number of adsorption sites
  per surface unit (1 to 5 per 1 nm).
• Typical surface coatings
• Normal phase (-Si-OH, -NH2)
• Reverse phase (C8, C18, Phenyl)
• Anion exchange (-NH4)
• Cation exchange (-COO-)

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Mobile Phase in HPLC

• Purity of the solvents
• Detector compatibility
• Solubility of the sample
• Low viscosity
• Chemical inertness
• Reasonable price

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Path of Mobile Phase
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Mobile phase degassing and storage

• It is recommended that you degas your solvents
  for several minutes before use (Helium) Special
  containers can prevent exchange with the ambient
  air (shown in this figure).
• This Waters 1525 HPLC is set up to do solvent
  gradients alternatively, you could premix the
  solvent and use one reservoir for isocratic runs.

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Mobile Phase mixing

• Solvent proportioning valves allow for gradient
  elution by being programmed to mix the solvents
  with respect to time

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HPLC Pump

• Reciprocating piston pumps are commonly used
  which have pistons that pull the mobile phase in
  and push it out into the head of the column

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Rotary Sample Loop Injector

• Injector needles are used ranging from 10 µL to
  500 µL to inject a sample onto the sample loop
• Upon a 60 rotation the pump introduces the
  sample onto the column in a reverse direction
  that it was loaded.

• http//www.restek.com/info_sixport.asp

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HPLC Columns

• HPLC Columns come in various sizes and many
  factors involving your analyte or the function of
  the column should be considered when selecting
  the appropriate one. Some common dimensions
  10, 15, and 25 cm in length 3, 5, or 10 mm
  diameters 4 to 4.6 internal diameters

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Column Cost and Sensitivity

• Costs generally range from 200 to 1000 per
  column.

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HPLC Detectors

• Most HPLC instruments are equipped with optical
  detectors.
• Light passes through a transparent low volume
  flow cell where the variation in light by UV
  Absorption, fluorescent emission, or change in
  refractive index are monitored and integrated to
  display Retention Time and Peak Area.
• Typical flow rates are 1 mL/min. and a flow cell
  volume of 5-50 µL.

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Common HPLC Detectors

• Refractive Index (RI) - universal
• Evaporative Light Scattering Detector (ELSD)
  universal
• UV/VIS light selective
• Fluorescence selective
• Electrochemical (ECD) selective
• Mass Spec (MS) - universal

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Refractive Index detector

• Analytes change the refractive index of the light
  in a proportional amount to the concentration.
• Heat can change the RI of the mobile phase so
  thermo control important
• RI changes cause a shift in a beams focal
  location which is detected on a photo-sensor.
• RI is ideal for analyzing complex sugars and
  carbohydrates which have no chromophores,
  fluorescence or electrochemical activities

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ELSD

• Light scatters in response to the dimension of
  the analyte particles.
• Light does not scatter in the mobile phase and
  must be nebulized and evaporated
• This universal detector is more sensitive that RI
  and shows a response to compound lacking UV
  absorption or fluorescence.
• Downfall is the sample is destroyed.

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UV/VIS Detectors

• Scan a range of UV light to detect molecules with
  chromophores. Commonly 254 nm.
• Usually having a range of 190 nm to 600 nm
• Low flow cell volume 1 10 µL
• Single wavelength filter photometers -uses a
  source lamp to emit a single wavelength (Hg, 254
  nm)
• Dispersive monochromator detectors -selects a
  narrow wavelength band
• Diode array detector -light from flow cell
  disperses and is directed towards different
  diodes

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Fluorescent Detectors

• Higher signal to noise ratio than UV/VIS
• Greater sensitivity than UV/VIS
• Many compounds do not fluoresce and are
  derivatized with chemicals such as Dansyl
  chloride. This works well with primary and
  secondary amines, amino acids and phenolic
  compounds.

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Electrochemical Detectors

• Selective detection commonly used with reverse
  phase and isocratic elution with buffers and
  salts as the mobile phase
• The two types of ECDs are voltammetric and
  conductometric
• The mobile phase must carry charged electrolytes
  eliminating normal phase as an option
• ECDs respond to analytes that are oxidizable or
  reducible at an electrode surface.

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Mass Spectrometer

• Problem interfacing the mobile phase with a MS
  detector
• The first interface system was a moving conveyer
  belt that passed through vacuum systems leaving
  the analyte on a solid adsorbent material
• Thermospray mobile phase is directed to a
  capillary column that is heated and points at a
  skimmer cone. (Too much build up on orifice)
• Electrospray (ESI) analytes are charged upon
  exiting the capillary tube and cross sprayed with
  nitrogen. The charge particles cause a Coulomb
  explosion making smaller droplets of analyte to
  enter the skimmer cone.
• Atmospheric Pressure Chemical Ionization (APCI)
  Analyte is heated by a ceramic tip on the column,
  cross flow of nitrogen decreases the droplet
  size, and a corona discharge charges the
  particles to enter the detector.

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Detector Summary
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Automated Waste Collection
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Typical Program Screen Waters software Breeze
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Why HPLC?

• HPLC works with compounds of higher molecular
  weights and polarity.
• Many biological samples are charged such as DNA
  and proteins.
• HPLC can be used in a prepatory manner with
  larger sample sizes and sample recovery to
  continue synthesis
• Good at separating stereoisomers techniques that
  employ heat (GC) can cause racemization during
  analysis.

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Contrasting HPLC and UPLC

• UPLC gives faster results with better resolution
• UPLC uses less of valuable solvents like
  acetonitrile which lowers cost
• The reduction of solvent use is more
  environmentally friendly
• Increased productivity can increase you revenue
  in an industrial setting

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Chromatograms of simvastatin
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Why is UPLC more efficient

• Peak capacity (P) is the number of peaks that can
  be resolved in a specific amount of time.
• P is proportional to the inverse of the square
  root of the Number of theoretical plates (N) N
  L/H
• Lower plate heights generate a smaller number of
  plates
• Plate heights are correlated through the Van
  Deemter equation

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Van Deemter Eqn.

• H A B/u Cu
• A is related to the mobile phase movement through
  paths in the stationary phase.
• B describes longitudinal diffusion
• C relates the analyte to mass transfer between
  the pores of the stationary phase
• Halasz eqn.

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Haslaz Eqn.

• Eqn.
• u relates to the velocity of the mobile phase
• dp depends on the particle size
• This formula implicates that decreasing particle
  size decreases the plate height which increases
  resolution.

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Synthetic ApplicationSemi-Prep
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Derivatization
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Typical Chiral Separation

• Biological activity depends on the
  stereochemistry of a particular enantiomer
• A common column is a cytodextrin packing with
  various glucopyranose units this column creates
  hydrophobic cavities with hydrophilic surfaces.
• The analyte is trapped in the cavity and can be
  separated from the polar solvents

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Quantitative Analysis Application

• HPLC can be use in conjunction with size
  exclusion to determine the molecular weight of
  proteins
• In this application molecules with larger weights
  have lower retention times.
• By plotting standard retention times in excel you
  can extrapolate the molecular weight (MW) of your
  protein
• Lactate Dehydrogenase MW analysis in an
  experiment was determined using a 280 nm
  wavelength with a TSKGel Super SW 3000 size
  exclusion 4.6mm 30 cm column, 50 mM phosphate
  buffer pH 7.2 containing 0.3 M NaCl as a mobile
  phase, a flow rate of 0.6 mL/min., 20 µL
  injection volume, and a Rheodyne injection valve.

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Standard protein molecular weight data
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References

• http//mtsu32.mtsu.edu11233/471toc.html
  (accessed 06/19/09).
• http//en.wikipedia.org/wiki/Chromatography
  (accessed 06/25/09).
• Robinson, J. W. Skelly Frame, E.M. Frame II,
  G.M. Undergraduate Instrumental Analysis, 6th
  ed. Marcel Dekker Inc. NY, 2005 pp 797-835.
• http//www.chem.queensu.ca/courses/08/CHEM321/Lect
  ureNotes/Chapter202520part20one.doc (accessed
  06/23/09).
• http//www.restek.com/info_sixport.asp (accessed
  06/20/09).
• http//www.waters.com/waters/promotionDetail.htm?i
  d10048693ev10007792localeen_US (accessed
  06/20/09).
• Dionex, Technical Note 75. Easy Method Transfer
  from HPLC to RSLC with the Dionex Method
  Speed-Up Calculator
• Levin, S. Abu-Lafi, S. The Role of
  Enantioselective Liquid Chromatography
  Separations Using Chiral Stationary Phases in
  Pharmaceutical Analysis, in Advances in
  Chromatography. Grushka, E. Brown, P. R.,
  Ed. Marcel Dekker Inc. NY, 1993 Vol. 33 pp
  233-236.
• Kline, P. Analysis of Lactate Dehydrogenase
  Determination of Molecular Weight and Purity,
  Middle Tennessee State University, Murfreesboro,
  TN, 2009.