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Gas chrmatography and HPLC

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Title: Gas chrmatography and HPLC


1
Gas ChromatographyCompiled By Million M.2016
2
  • 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

3
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

4
  • 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

5
  • 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 .

6
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

7
Instruments for GC
8
Main components of GC
  • Carrier gas (mobile phase)
  • Sample injection port
  • Column
  • Oven
  • Detectors

9
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.

10
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)

11
  • 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.

12
Many based on polysiloxanes or polyethylene
glycol (PEG)
13
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.

14
  • 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
15
  • 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.
16
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
17
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.

18
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.
19
Open Tubular Column for Gas Chromatography
Fused silica tubing
Gaseous Mobile phase
dc
Stationary phase
20
  • 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.

21
The performance of SCOT is intermediate between
those of WCOT and packed columns.
22
(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.
23
  • 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)

24
  • 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
25
  • 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

26
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.

27
  • 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.

28
  • Thermal-conduc. (TCD) and flame ionization (FID)
    detectors are the two most common detectors on
    commercial GCs.

29
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

30
  • 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)

31
  • 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.

32
  • 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.

33
  • 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.

34
  • 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.

35
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.

36
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.
37
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.

38
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.
39
Separation in GC
Analyte
To detector
Column packing
Time
40
Separations
To detector
41
  • 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).

42
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

43
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.

44
  • 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

45
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

46
  • 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

47
  • 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

48
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?
49
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.
50
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?
51
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.
52
Exercise 3
Consider the following compounds
If they are run on an DEGS GC column, which
column will have the lowest RT? Highest?
53
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.
54
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?

55
  • 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?

56
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.

57
High-performance Liquid Chromatography (HPLC)
58
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

59
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

60
  • 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

61
Instrumentation
  • Mobile Phase Reservoirs
  • Pumps
  • Sample injection system
  • Column
  • Detectors
  • Recorders and Integrators

62
(No Transcript)
63
High-Performance Liquid Chromatography (HPLC)
64
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

65
  • 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.

66
  • Separation efficiency is greatly enhanced by
    gradient elution. See on the following peaks

67
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)

68
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

69
  • 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

70
The following table shows the polarity indexs of
different mobile phases
71
  • 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

72
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

73
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

74
The Load position and Inject mode
75
  • 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
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