Gas chrmatography and HPLC

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Gas ChromatographyCompiled By Million M.2016
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• Objectives
• Define Gas Chromatography (GC) and distinguish
  between GSC and GLC.
• Describe different types of columns and
  stationary phases employed in GC, and explain
  their use
• Distinguish between split, split-less, and
  on-column injection techniques.
• Describe the function and usage of common
  detectors in GC.
• Outline the fundamental basis for separation in
  GC
• Indicate the major advantages of GC and the
  application areas in which it is used

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Gas Chromatography (GC)

• Gas chromatography is a chromatographic technique
  that can be used to separate thermally stable and
  volatile organic and inorganic compounds.
• In gas chromatography the sample is vaporized and
  injected on to the head of a chromatographic
  column
• Where the components of a vaporized samples are
  separated by being distributed between a mobile
  gaseous phase and a liquid or a solid stationary
  phase held in a column

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• Types of GC
• Two types of gas chromatography
• 1. Gas-solid chromatography (GSC)
• GSC is based up on adsorption of gaseous
  substances on solid surfaces.
• Common solid stationary phases are silica gel,
  molecular sieves, porous polymers, alumina and
  charcoal
• Distribution of coefficients are generally much
  larger than for those GLC.
• Consequence GSC is use full for separation of
  species that are not retained by GLC columns,
  such as
• the components of air, hydrogen sulfide,
  nitrogen oxides,
  carbon monoxide, carbon dioxide and the
  rare gases

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• GSC has limited application owing to the semi
  permanent retention of active or polar molecules
  and consequence of the nonlinear character of
  adsorption process.
• Thus, the technique has not found wide
  application except for the separation of
  low-molecular weight gaseous species as mentioned
  above .

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2. Gas-liquid chromatography (GLC)

• GLC finds wide spread of use in all fields of
  science, where its name is usually shortened to
  GC
• Gas-liquid chromatography is based upon the
  partition of the analyte between a gaseous mobile
  phase and a liquid phase immobilized on the
  surface of an inert solid.
• The liquid is spread (coated) as thin film over
  an inert support

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Instruments for GC
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Main components of GC

• Carrier gas (mobile phase)
• Sample injection port
• Column
• Oven
• Detectors

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Carrier Gases (mobile phase)

• The Carrier gases
• Must be chemically inert toward both the sample
  and stationary phase, includes He2, N2, Ar and
  H2
• Must be dry and free of impurities
• Must be compatible to the detector
• In addition, the carrier gas system often
  contains a molecular sieve to remove water or
  other impurities.
• He2 is the most common gas because it is
  compatible with all detectors.

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Stationary Phases

• Selectivity in GC is influenced by the choice of
  stationary phase.
• Elution order in GLC is determined primarily by
  the solutes boiling point and to a lesser
  degree, by the solutes interaction with the
  stationary phase.
• Solutes with significantly different boiling
  points are easily separated
• On the other hand, two solutes with similar
  boiling points can be separated only if the
  stationary phase selectively interacts with one
  of the solutes.
• In general, nonpolar solutes are more easily
  separated with a nonpolar stationary phase, and
  polar solutes are easier to separate using a
  polar stationary phase( in a similar way with
  like dissolves like)

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• The main criteria for selecting a stationary
  phase for gas-liquid chromatography
• it should be of low volatility (ideally, the
  boiling point of the liquid should be at least
  100 oC higher than the maximum operating
  temperature for the column)
• it should be chemically inert,
• thermally stable, and of an appropriate polarity
  for the solutes being separated.

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Many based on polysiloxanes or polyethylene
glycol (PEG)
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Sample Injection System
To avoid any pre-column loss in resolution due to
band broadening, a sample of sufficient size must
be introduced in a small volume of mobile phase

• Colmun efficiency requires that the sample to be
  of suitable size
• Slow injection of over sized samples causes band
  spreading and poor resolution.
• The most common method of sample injection
  involves the use of micro syringe to inject a
  liquid or gaseous sample through a self-sealing
  silicon rubber.

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• The sample port is maintained at a higher
  temperature than the boiling point of the least
  volatile component in the sample mixture (50 oC
  above the boiling point of the least volatile
  component of the sample)
• Capillary columns require the use of a special
  injector to avoid overloading the column with
  sample. Therefore, several capillary injectors
  are available, but the most commons are

Common injection techniques 1) Hot flash
vaporization Split Splitless 2) Direct cold
on column Split or on-column Split
1) Simple 2) High column efficiency 3)
Column may be protected On-column 1) Best
accuracy 2) Thermolabile compounds 3) Trace
analysis
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• Split injection A technique for injecting
  samples onto a capillary column in which only a
  small portion of the sample enters the column
  (0.1-1 injected sample reaches the detcetor)
• Because of the limited capacity for sample of
  these small bore columns and the difficulty of
  injecting extremely small volume samples, a
  large portion of the injected sample is vented
  to atmosphere by the inlet.

Representative injection conditions for split,
splitless, and on-column injection into an open
tubular column.
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Splitless injection A technique for injecting a
sample onto a capillary column that allows a
higher percentage of the sample to enter the
column. (used for trace analysis) On-column
injection the direct injection of thermally
unstable samples onto a capillary column
Disadvantage of splitless 1) Slow sample
transfer to column 2) Must dilute sample with
volatile solvent 3) Time consuming must
cool column 4) Poor for thermolabile compounds

• Advantage of on-column
• 1) Best reproducibility Quantitative results
• 2) No split, no loss of high boilers
• 3) "Cold" on-column injection available
• Advantage of splitless
• High sensitivity ( 95 of sample on column)
• Solvent effect produces narrow sample bands
• 3) Same hardware as split injection

Split Injection Disadvantages 1.Not suitable for
ultra-trace analysis 2.Suffers from
Discrimination 3. Analytes susceptible to thermal
degradation
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GC Columns
Capillary ( open tubular) columns
Packed columns

• Typically a glass or stainless steel coil.
• 1-5 m total length and 3-6 mm inner diameter.
• Filled with the st. ph. or a packing coated with
  the st.ph.
• used for preparative separations

• Thin fused-silica.
• Typically 10-100 m in length and 0.1-0.7 mm inner
  diameter.
• St. ph. coated on the inner surface.
• Provide much higher separation efficiency
• But more easily overloaded by too much sample.

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Packed Column
Commonly used for gas or liquid chromatography
Gas or Liquid Mobile phase
dc
Packing (stationary phase)
Packed columns contain fine particles of solid
support coated with nonvolatile liquid stationary
phase, or the solid itself may be the stationary
phase. Compared with open tubular columns,
packed columns provide greater sample capacity
but give broader peaks, longer retention times,
and less resolution.
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Open Tubular Column for Gas Chromatography
Fused silica tubing
Gaseous Mobile phase
dc
Stationary phase
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• Types of open tubular Columns
• Open tubular, or capillary, columns are of two
  basic types,
• Wallcoated open tubular (WCOT) and
• Support-coated open tubular (SCOT).
• The wall-coated column features a 0.1- to
  5-?m-thick film of stationary liquid phase on the
  inner wall of the column
• A support-coated column has solid particles
  coated with stationary liquid phase and attached
  to the inner wall.
• With their high surface area, SCOT can handle
  larger samples than WCOT.
• In the porous-layer column in solid particles are
  the active stationary phase.

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The performance of SCOT is intermediate between
those of WCOT and packed columns.
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(Left) Gas chromatogram of alcohol mixture at 40
oC using packed column ( 2mm I.D., 76 cm long
containing 20 Carbowax 20 M on a Gas-Chrom R
support and FID. (Right) Chromatogram of vapors
from headspace of beer can, obtained with 0.25 mm
diameter, 30 m long porous carbon column oerated
at 30 oC for 2 min and then ramped up to 160 oC
at 20 oC/min.
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• Column temperature is an important variable must
  be controlled
• Thus, the column is ordinarily housed in
  thermostated oven.
• The optimum column temperature depends up on the
  boiling point of the sample and the degree of
  the separation required
• Roughly a temperature equal to or slightly above
  the average boiling point of a sample results in
  a reasonable elution time (2 to 30 min)

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• With a broad boiling ranges it is often desirable
  to employ temperature programming, whereby the
  column temperature is increased continuously or
  in steps as the separation proceeds.
• Resolution is associated with minimal temperature
  , but as temp. decrease, increase in elution time
  therefore the time required to complete the
  analysis will become long

Modes of Separation GC Isothermal constant
temperature Gradient varying temperature
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• This principle is illustrated by the following
  peaks.
• As column temperature raised, vapor pressure
  analyte increases, eluted faster.
• Raise column temperature during separation
  temperature programming separates species with
  wide range of polarities or vapor pressures

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GC Detectors

• Detectors are used to generate analytical signal
  
• Characteristics of the Ideal Detector The ideal
  detector for gas chromatography has the following
  characteristics
• 1. Adequate sensitivity
• 2. Good stability and reproducibility.
• 3. A linear response to solutes that extends over
  several orders of magnitude.
• 4. A temperature range from room temperature to
  at least 400oC.

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• 5. A short response time that is independent of
  flow rate.
• 6. High reliability and ease of use.
• 7. Similarity in response toward all solutes or a
  highly selective response toward one or more
  classes of solutes.
• 8. Nondestructive of sample.

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• Thermal-conduc. (TCD) and flame ionization (FID)
  detectors are the two most common detectors on
  commercial GCs.

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Flame Ionization Detectors (FID)

• Principle
• GC effluent burned in hydrogen flame (CH/CHO
  radicals)
• Ions produced in combustion and collected in
  electrode
• Current proportional to mass combusted

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• Most carbon atoms, except those in carbonyl and
  carboxylic groups, generate a signal, making the
  FID an almost universal detector for organic
  compounds
• The FID exhibits a high sensitivity, large linear
  response range (107), and low noise

• Advantages
• It is very sensitive for most organic compounds
  (1 pg/s)
• Low sensitivity for small molecules i.e., N2, CO,
  CO2, H2O
• Disadvantages
• The sample is destroyed ?
• It requires three gases (carrier gas (i.e.,
  helium, argon, nitrogen), hydrogen and
  air/oxygen)

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• Thermal Conductivity Detectors(TCD)
• A very early detector for gas chromatography,
• And still it has wide application,
• Principle
• heat conducted by gas from heated filament
• Filament resistance is temperature dependent
• Heat conduction depends on gas MW
• All gases (w/ MW ? carrier MW) detected, but
  variable sensitivity.

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• The advantage of the TCD is
• its simplicity,
• its large linear dynamic range(105),
• its general response to both organic and
  inorganic species,
• its nondestructive character, which permits
  collection of solutes after detection.
• A limitation of this detector is its relatively
  low sensitivity (10-8 g solute/mL carrier gas).
• Other detectors exceed this sensitivity by
  factors as large as 104 to 107.

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• Electron-Capture Detectors(ECD)
• Principle
• The Uses ß emitter to produce electrons that
  cause current
• Compounds with electronegative elements (e.g.
  halogens) capture electrons reducing current
• Ni-63 ? e- N2 ? 2e- N2
• One of the most sensitive detectors available
• ECD is selective in its response to molecules
  containing electronegative functional groups such
  as halogens, peroxides, quinones, and nitro
  groups.

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• It is insensitive to functional groups such as
  amines, alcohols, and hydrocarbons.
• An important application of the ECD has been for
  the detection and determination of chlorinated
  insecticides.
• Advantages
• It is very sensitive for chlorinated compounds
  i.e., Tetrachlorodibenzo-p-dioxin (TCDD),
  Polychlorinated biphenyls (PCB), etc.
• Disadvantages
• It requires a radioactive source and special
  license to operate these sources!
• Several carrier gases needed for the ionization
  i.e., argon/methane.

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Coupling a Gas Chromatogram to a Mass
Spectrometer (GC-MS)

• A is an instrument that ionizes a gaseous
  molecule using enough energy that the resulting
  ion breaks apart into smaller ions.
• Because these ions have different mass-to-charge
  ratios, it is possible to separate them using a
  magnetic field or an electrical field.
• The resulting mass spectrum contains both
  quantitative and qualitative information about
  the analyte.

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Mass spectrum for toluene highlighting the
molecular ion in green (m/z 92),
Block diagram of GCMS. A three component mixture
enters the GC. When component A elutes from the
column, it enters the MS ion source and ionizes
to form the parent ion and several fragment ions.
the height of any peak is proportional to the
amount of toluene in the mass spectrometer and
the fragmentation pattern is unique to toluene.
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Separation in GC

• Different compounds have different retention
  times. For a particular compound, the retention
  time will vary depending on
• The boiling point of the compound.
• A compound which boils at a temperature higher
  than the column temperature is going to spend
  nearly all of its time condensed as a liquid at
  the beginning of the column.
• So high boiling point means a long retention time.

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The solubility in the liquid phase. The more
soluble a compound is in the liquid phase, the
less time it will spend being carried along by
the gas. High solubility in the liquid phase
means a high retention time. The temperature of
the column. A higher temperature will tend to
excite molecules into the gas phase - because
they evaporate more readily.
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Separation in GC
Analyte
To detector
Column packing
Time
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Separations
To detector
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• Chemical Derivatization (prior to analysis)
• GC not always possible (biomedical and
  environmental interest) particularly for those of
  high molecular weight and/or molecules containing
  polar functional groups.
• Derivatization used when analytes are not
  sufficiently volatile, tail significantly (too
  strongly attracted to the stationary phase) and
  thermally unstable (decompose).

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Application of gas chromatography

• Gas chromatography is widely used for the
  analysis of a diverse array of samples in
• In environmental samples for the analysis of
  numerous organic pollutants in air, water, and
  wastewater
• In clinical, pharmaceutical, and forensic labs
  make frequent use of gas chromatography for the
  analysis of drugs and monitoring blood alcohol
  levels
• In food science consumer goods, many flavors,
  spices, and fragrances are readily analyzed by
  GC, and
• In petrochemical laboratories GC is ideally
  suited for the analysis of petroleum products,
  including gasoline, diesel fuel, and oil

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Qualitative Analysis

• 1. To confirm purity of organic cpds
• GC are widely used as criteria of purity for
  organic compounds.
• Contaminants, if present, are revealed by the
  appearance of additional peaks the areas under
  these peaks provide rough estimates of the extent
  of contamination.
• The technique is also useful for evaluating the
  effectiveness of purification procedures.
• 2.To identify the cpds by their retention time
• Retention times should be useful for the
  identification of components in mixtures.

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• Sine the GC gives a single piece of information
  about each species in a mixture (the retention
  time), the application of the technique to the
  qualitative analysis of complex samples of
  unknown composition is limited.
• This limitation has been largely overcome by
  linking chromatographic columns directly with
  ultraviolet, infrared, and mass spectrometers to
  produce hyphenated instruments

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Quantitative Analysis

• In GC quantitative analysis can be carried out
  using either
• Peak area or
• Peak height
• Abetter choice is to measure the area under the
  chromatographic peak with an integrating recorder
• Since peak area is directly proportional to the
  amount of analyte that was injected, changes in
  column efficiency will not affect the accuracy or
  precision of the analysis unlike the peak height
• Calibration curves are usually constructed by
  analyzing a series of external standards and
  plotting the detectors signal as a function of
  their known concentrations

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• GC Advantages
• Fast analysis
• High efficiency leading to high resolution
• Sensitive detectors (ppb)
• Non-destructive enabling coupling to Mass
  Spectrometers (MS) - an instrument that measures
  the masses of individual molecules that have been
  converted into ions, i.e. molecules that have
  been electrically charged
• High quantitative accuracy (lt1 RSD typical)
• Requires small samples (lt1 mL)
• Rugged and reliable techniques

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• GC Disadvantages
• Limited to volatile samples
• Not suitable for samples that degrade at elevated
  temperatures (thermally labile)
• Not suited to preparative chromatography
• Requires MS detector for analyte structural
  elucidation (characterization)
• Most non-MS detectors are destructive

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Exercise 1
Study the chromatograph (below) of a mixture of
Compounds A and B, run on the GCs. Compound A has
the shorter retention time.
1. What is the retention time of compound A?
Compound B? 2. Which compound is present in a
larger amount? 3. Which compound has the lower
boiling point? 4. What would happen to the
retention times of compounds A and B if the
column temperature were raised?
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Answer
1. The retention time of compound A is 1.11. this
number is read both at the top of the peak and in
the RT column. The retention time of compound B
is 2.27. 2. Compound A is present is a larger
amount, since it makes up 56 of the mixture. 3.
Compound A has the lower boiling point since it
comes off the column first. 4. If the column
temperature were raised, both compounds A and B
would boil sooner and thus come off the column in
a shorter time. Thus, the retention times of both
compounds A and B would be smaller than before
the temperature was raised.
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Exercise 2

• Consider the following compounds

If the compounds in the table above are run on an
OV-101 GC column, which compound will have the
lowest RT? Highest?
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Answer OV-101 GC columns are the ones used in
the Gow-Mac columns they are non-polar and
separate essentially on boiling point, especially
if the compounds are similar in structure. All
of the compounds in the table are hydrocarbons,
hence of similar structure. The one with the
lowest boiling point, pentane, will have the
lowest RT. The compound with the largest
boiling point, 2,3-dimethyl octane, will have the
highest RT.
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Exercise 3
Consider the following compounds
If they are run on an DEGS GC column, which
column will have the lowest RT? Highest?
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Answer
DEGS columns are much more polar than OV-101
columns. When using DEGS columns, the polarity of
the samples will have an effect on the rate of
their movement through the polar column polar
compounds will be retained longer than non-polar
compounds, especially if the boiling points are
similar. Of the compounds in the table (note
that they all have similar boiling points),
propionic acid is the most polar, and hence will
be retained the longest and have the largest RT.
3,4-dimethylheptane is the least polar, and
therefore will have the lowest RT.
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Questions

1. List two ways to inject gas samples on GCs set up
   for analysis of liquids.
2. For what type of columns can direct injection be
   used?
3. What does the retention time of a species tell
   you about the compound? For example If you have
   a chromatogram with two peaks corresponding to C1
   C2, then what does it mean for tR1 to be less
   than tR2?
4. What are the desirable characteristics of a GC
   detector ?
5. What do you understand by temperature programming
   in GC analysis?
6. Derivatisation of a sample is carried out in GC
   to do what?

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• 7. If Compound A has a boiling point of 35ºC,
  Compound B has a boiling point of 85ºC and
  Compound C has a boiling point of 133ºC, which of
  the compounds will come through the GC first?
  Last?
• 8. What does the area under the peak on a GC tell
  you?

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References

• Principles of instrumental analysis, 5th ed. by
  Skoog, Holler, Nieman Chapter 27.
• Quantitative Chemical Analysis, 7th ed. By
  Harris Chapter 24.
• Lecture of Chromatography-III by Dr. Raimund
  Niess, GUC, 2009.

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High-performance Liquid Chromatography (HPLC)
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HPLC

• H High
• P Performance/Pressure
• L Liquid
• C Chromatography
• HPLC is the most widely used of all the
  analytical separation techniques.
• The reasons for the popularity of the method is
  its
• sensitivity
• its ready adaptability to accurate quantitative
  determinations
• Its suitability for separating nonvolatile
  species or thermally fragile ones

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TYPES OF HPLC TECHNIQUES

• Based on modes of chromatography
• 1. Normal phase mode
• 2.Reverse phase mode
• B. Based on principle of separation
•  1. Liquid/Solid Chromatography
  (adsorption chromatography)
•  2. Liquid/Liquid Chromatography (partition
  chromatography)
•  3. Ion Exchange Chromatography
•  4. Gel Permeation (size exclusion )
  chromatography 5. Affinity chromatography

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• C. Based on the scale of operation
• 1. Analytical HPLC
• 2. Preparative HPLC
• D. Based on elution technique
• 1. Isocratic elution HPLC
• 2. Gradient elution HPLC

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Instrumentation

• Mobile Phase Reservoirs
• Pumps
• Sample injection system
• Column
• Detectors
• Recorders and Integrators

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High-Performance Liquid Chromatography (HPLC)
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Mobile Phase Reservoirs and SolventTreatment
Systems

• Should be Inert container with inert lines
  leading to the pump
• Reservoir filters (2-10 mm) at reservoir end of
  solvent delivery lines
• The reservoirs are often equipped with a means of
  removing dissolved gases such as oxygen and
  nitrogen that interfere by forming bubbles in the
  column and the detector.
• Degassed solvent the dissolved gasses can be
  removed using
• - Vacuum filtration
• - Sparge with inert gas (N2 or He)
• - Ultrasonic under vacuum

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• Use a mobile phase (kinds of elution)
• Isocratic elution A separation that employs a
  single solvent or solvent mixture of constant
  composition.
• Can only use one pumps
• Mix solvents together ahead of time
• Simpler, no mixing chamber required
• Limited flexibility, not used much in research,
  mostly process chemistry or routine analysis
• Gradient elution In gradient elution, two (and
  sometimes more) solvent
• systems that differ significantly in polarity are
  used and varied in composition
• during the separation
• Use multiple pumps whose out put is mixed
  together
• After elution is begun the ratio of the solvents
  is varied in a programmed way, sometimes
  continuously and sometimes in a series of steps.

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• Separation efficiency is greatly enhanced by
  gradient elution. See on the following peaks

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Requirements of mobile phase in HPLC.

• Must be
• Solvate the analyte molecules and the solvent
  they are in
• Be suitable for the analyte to transfer back and
  forth b/n during the separation process
• Compatible with instrument (pumps, seals,
  fittings, detectors etc)
• Compatible with the stationary phase
• Readily available
• Of adequate purity
• Not too compressible (causes pumps/flow problems)
• Free of gases (which cause compress ability
  problems)

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Selection of mobile phase (solvent)

• The liquid stationary mobile phases should have
  a considerable difference between their solvent
  strength parameters.
• Polarity strength of solvents
• Pure water gt Methanol gt Ethanol gt Propanol gt
  Acetone gt Ethyl acetategt Ether gt Chloroform gt
  Dichloromethane gtBenzene gt Toluene gt Carbon
  tetrachloride gt Cyclohexane gt Hexane gt Pentane.
• e.g. if the stationary phase is water, pentane
  would be the eluent of choice.
• i.e. if the stationary phase is polar the mobile
  phase should be non polar/slightly polar and vice
  versa

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• Several indices have been developed to assist in
  selecting a mobile phase, the most useful of
  which is the polarity index
• Provides values for the polarity index, P of
  several commonly used mobile phases, in which
  larger values of P correspond to more polar
  solvents.
• Mobile phases of intermediate polarity can be
  fashioned by mixing together two or more of the
  mobile phases
• For example, a binary mobile phase made by
  combining solvents A and B has a polarity index,
  PAB of

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The following table shows the polarity indexs of
different mobile phases
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• Example
• A reverse-phase HPLC separation is carried out
  using a mobile-phase mixture of 60 v/v water and
  40 v/v methanol. What is the mobile phases
  polarity index?
• Solution From Table 12.3 we find that the
  polarity index is 10.2 for water and 5.1 for
  methanol. Using the above equation, the polarity
  index for a 6040 watermethanol mixture is
• PAB (0.60)(10.2)
  (0.40)(5.1) 8.2

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Pumps

• Is used to force the mobile phase through the
  column
• The requirements for liquid chromatographic pumps
  include
• 1. the generation of pressures of up to
  6000 psi (lb/in2),
• 2. pulse-free output,
• 3. flow rates ranging from 0.1 to 10
  mL/min,
• 4. flow reproducibilities of 0.5
  relative or better, and
• 5. resistance to corrosion by a
  variety of solvents
• Different types of pumps
• Reciprocating type pumps is used in almost all
  commercial instrument
• Syringe type pumps
• Constant pressure pumps

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Sample injection

• Several devices are available either for manual
  or auto injection of the sample
• Different devices are 1.Septum injectors
• 2. Stop flow injectors
• 3. loop valve type
  (Rheodyne injectors )
• Loop valve injector A means for injecting
  samples in which the sample is loaded into a
  short section of tubing and injected onto the
  column by redirecting the mobile phase through
  the loop
• Is the most popular injector.
• This has a fixed volume loop like 20 µl or 50 µl
  or more.
• The Injector has 2 modes. Load position and
  Inject mode.
• Sample size 0.5 500 µL
• No interference with the pressure
• P lt 7000 psi
• Auto sampler inject continuously variable volume
  1 µL 1 mL

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The Load position and Inject mode
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• Analytical Columns is the heart of separation
• Liquid-chromatographic columns range in length
  from 3 to 7.5 cm.
• Normally, the columns are straight, with added
  length, where needed, being gained by coupling
  two or more columns together.
• The inside diameter of liquid columns is often 1
  to 4.6 mm and
• The most common particle size of packings is 3 or
  5 ?m.
• Columns of this type contains as many as 100,000
  plates/meter

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Column in Liquid Chromatography
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• Guard Columns
• A guard column is introduced before the
  analytical column to increase the life of the
  analytical column by removing
• particulate matter and contaminants from the
  solvents
• sample components that bind irreversibly to the
  stationary phase.
• The guard column serves to saturate the mobile
  phase with the stationary phase so that losses of
  this solvent from the analytical column are
  minimized.
• Its packing composition is similar to that of the
  analytical column the particle size is usually
  larger.
• When the guard column has become contaminated, it
  is repacked or discarded and replaced with a new
  one.

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• DETECTORS
• Detectors used depends upon the property of the
  compounds to be separated.
• The ideal detector for HPLC should have all the
  characteristics of the ideal GC detector
• except that it need not have as great a
  temperature range.
• In addition, an HPLC detector must have low
  internal volume (dead volume) to minimize
  extra-column band broadening.
• UV detector is the most common detector in HPLC

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Partition Chromatography

• Partition chromatography has become the most
  widely used of all the types of LC
• two types
• (i) liquid-liquid chromatography and
• (ii) bonded-phase chromatography.
• With liquid-liquid, a liquid stationary phase is
  retained on the surface of the packing by
  physical adsorption.
• With bonded-phase, the stationary phase is bonded
  chemically to the support surfaces.

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• Bonded-phase method has become predominate
  because of certain disadvantages of liquid-liquid
  systems.
• The disadvantages of liquid-liqiud is
• loss of stationary phase by dissolution in the
  mobile phase, which requires periodic recoating
  of the support particles.
• Furthermore, stationary-phase solubility problems
  prohibit the use of liquid-phase packings for
  gradient elution.

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• Both liquid liquid and bonded-phase
  chromatography's classified in to two based up
  on the relative polarities of mobile and
  stationary phase
• 1. Normal Phase
• Stationary phase Polar with short carbon
  chains
• Mobile phase Non-polar such as hexane
• The least polar component is eluted
  first increasing the polarity of the mobile
  phase then decreases the elution time
• 2. Reverse Phase is more common
• Stationary Phase Non polar (Hydrophobic
  long chain C)
• Mobile Phase Polar usually aqueous (eg.
  Methanol (CH3OH), acetonitrile (CH3CN),
  tetrahdofuran, water
• The most polar component elutes first, and
  increasing the mobile phase polarity increases
  the elution time

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Adsorption chromatography

• Adsorption, or liquid-solid is classic form of
  liquid chromatography first introduced by Tswett
• The only stationary phases that are used for
  liquid-solid HPLC are silica and alumina
• Since the stationary phase is polar, the mobile
  phase is usually a nonpolar
• or moderately polar solvent
• Typical mobile phases include hexane, isooctane,
  and methylene chloride.
• The usual order of elution, from shorter to
  longer retention times, is
• olefins lt aromatic hydrocarbons lt ethers lt
  esters, aldehydes, ketones
• lt alcohols, amines lt amides lt carboxylic acids

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• Liquid solid chromatography is best suited to
  samples that are soluble in nonpolar solvents and
  correspondingly have limited solubility in
  aqueous solvents such as those used in the
  reversed phase partition procedure
• Separation of isomers is also usually better with
  the adsorption procedure

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Ion-Exchange Chromatography(IC)

• Ion-exchange chromatography is often shortened to
  ion chromatography
• Is an efficient methods of separating and
  determining ions based on ion-exchange resins
• Is used to separate charged species
• The mobile phase in IEC is usually an aqueous
  buffer,
• Then the pH and ionic composition of which
  determines a solutes retention time

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• Principle
• 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" (Lattice or framework)
  that carries fixed charges (not exchangeable) and
  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 can be replaced by ions
  of the same sign present in the surrounding
  solution.
• The "matrix" (framework) of a "cation exchanger"
  is considered as a crystalline non-ionized
  "polyanion" the matrix of an "anion exchanger"
  as a non-ionized "polycation"

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Ion Exchangers
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Cation Exchange Chromatography

• Cation exchange chromatography retains positively
  charged cations because the stationary phase
  displays a negatively charged functional group
• The active ions (counter ions) are cation.
• For cation separation the cation-exchange resin
  is usually acidic i.e.
• The polar groups attached to the matrix are
  acidic (sulphonic acids, carboxylic acids,
  phenols, phosphoric acids) e.g. a cation
  exchanger in the free carboxylic acid form

• X-COO- H
• X Frame work (matrix)
• -COO- Fixed charge (anionic),
• Non-exchangeable
• H Counter ion (cation), Exchangeable

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• Example The ion-exchange reaction of a
  monovalent cation, M, at a strong acid exchange
  site is

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Anion Exchange Chromatography

• Anion exchange chromatography retains anions
  using positively charged functional group
• The active ions (counter ions) are anions.
• The polar groups attached to the matrix are
  tertiary or quaternary ammonium groups (basic).
• e.g. Anion exchanger in the quaternary ammonium
  form
• X. NR3OH
• X Framework (matrix)
• -NR3 Fixed charge (cationic)
• Non exchangeable
• -OH counter ion (anion), Exchangeable

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IC Instrumentation
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Applications of Ion Exchange Chromatography

• 1- Water softening
• Removal of Ca2, Mg2 other multivalent ions
  causing hardness of water by filtration through a
  layer of strong cation resin.
• 2-Water demineralization Removal of cations
  anions dissolved in water.
• 3- Neutralization Cationic exchanger in H can
  be used to neutralize alkali hydroxide anionic
  exchanger in OH- form to neutralize the acidity
• 4- Separation of electrolytes from
  non-electrolytes
• 5- Separation of carbohydrates their
  derivatives

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Size Exclusion Chromatography (SEC)

• It is known as gel permeation or gel filtration
  chromatography
• SEC is a high performance liquid chromatography
  technique for the separation of components based
  on their size (diameter) and in some cases
  molecular weight of the target molecule in
  question

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Principle

• There is a distribution of pore sizes within the
  stationary phase such that the small molecules
  can enter most of the pores and are therefore
  retained the longest, while the larger molecules
  enter fewer pores and are retained a shorter time
  period.
• The underlying principle of SEC is that particles
  of different sizes will elute (filter) through a
  stationary phase at different rates.
• Particles of the same size should elute together
• Mobile Phase solvent plus portion of the
  polymer (solvent molecule to be separated )
• Stationary phase the porous material
  like silica particles and
• cross-linked polymer resin beads

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Principle
Ref L. H. Sperling, Introduction to Physical
Polymer Science, John WileySons Inc. Bethlehem,
Pennsylvania 4th Ed.2005
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• Column Packings Two types of packing for
  size-exclusion chromatography are encountered
• polymer beads and
• silica-based particles, both of which have
  diameters of 5 to 10
• Numerous size-exclusion packings are on the
  market
• Some are hydrophilic for use with aqueous mobile
  phases others are hydrophobic and are used with
  nonpolar organic solvents
• Gel filtration is a type of size exclusion
  chromatography in which the packing is
  hydrophilic and is used to separate polar species
• Gel permeation is a type of size-exclusion
  chromatography in which the packing is
  hydrophobic and is used to separate nonpolar
  species

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Instrumentation and Structure
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• The collected fractions are often examined by
  spectroscopic techniques to determine the
  concentration of the particles eluted
• Common spectroscopy detection techniques are
  refractive index (RI) and ultraviolet (UV)
• SEC is a widely used technique for the
  purification and analysis of synthetic and
  biological polymers, such as proteins,
  polysaccharides and nucleic acids
• SEC is used for the determination of the
  molecular mass
• A calibration curve of log molecular weight
  versus retention volume plot can be used to
  estimate the molecular weight

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Fig. 5. Theoretical chromatogram of a high
resolution fractionation (UV
absorbance)
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Affinity Chromatography

• In affinity chromatography, a reagent called an
  affinity ligand is covalently bonded to a solid
  support
• Typical affinity ligands are antibodies, enzyme
  inhibitors, or other molecules that reversibly
  and selectively bind to analyte molecules in the
  sample.
• When the sample passes through the column, only
  the molecules that selectively bind to the
  affinity ligand are retained.
• Molecules that do not bind pass through the
  column with the mobile phase.
• After the undesired molecules are removed, the
  retained analytes can be eluted by changing the
  mobile phase conditions

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• The stationary phase for affinity chromatography
  is a solid, such as agar ose, or a porous glass
  bead to which the affinity ligand is immobilized
• The role of mobile phase in affinity
  chromatography
• First, it must support the strong binding of the
  analyte molecules to the ligand.
• Second, once the undesired species are removed,
  the mobile phase must weaken or eliminate the
  analyte-ligand interaction so that the analyte
  can be eluted
• The primary use is in the rapid isolation of
  biomolecules during preparative work.

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Summery of the Liquid Chromatography