Introduction to chromatography tecniques

1
Instrumental Analysis I
Chem. 2051
Million M. 2016
2
Analytical separation techniques and classical
method of analysis

• Definition of Analytical chemistry deals with
  the methods used to determine the chemical
  composition of a sample of matter.
• Chemical composition is determined
• qualitatively answers what is present? and
• quantitatively answers how much each
  component is present?
• Analytical methods of analysis
• A. Classical(wet-chemical methods)
• Separation of analytes - extraction,
  distillation, precipitation (precipitation),
  filtration (filtering), etc..
• Qualitative Analysis - boiling point, freezing
  point, color, odor, density, reactivity,
  refractive index, etc..

3

• 3. Quantitative Analysis - gravimetric and
  volumetric analysis
• B. Instrumental Methods of Analysisexploit the
  physical properties of an analyte to obtain
  information, both qualitative and quantitative.
• 1. Separation of analytes- Can be done in 2
  waysa. Physical separation -
  Chromatography - Electrophoresisb.
  Spectroscopic separation isolate the signal
  that appears in spectroscopy

4

• 2. Qualitative Analysis
• X-Ray Spectroscopy
• Infrared Spectroscopy (IR)
• mass spectroscopy (MS)
• nuclear magnetic spectroscopy (NMR)
• 3. Quantitative Analysis
• UV-Vis Spectroscopy
•  Atomic absorption emission spectroscopy
  (AAS and AES)
• mass spectroscopy (MS)

5
Classifying Separation Techniques
6
General Theory of Separation Efficiency

• The goal of an analytical separation is to remove
  either the analyte or the interferent from the
  sample matrix.
• To achieve a separation there must be at least
  one significant difference between the chemical
  or physical properties of the analyte and
  interferent.
• Here selectivity is also very important
• A separations efficiency is influenced both by
  the failure to recover all the analyte and the
  failure to remove all the interferent.
• We define the analytes recovery, RA, as

7

• where CA is the concentration of analyte
  remaining after the separation,
• and (CA)o is the
  analytes initial concentration.
• A recovery of 1.00 means that none of the analyte
  is lost during the separation.
• The recovery of the interferent, RI, is defined
  in the same manner
• where CI is the concentration of interferent
  remaining after the separation, and
• (CI)o is the interferents initial concentration.

8

• The degree of separation is given by a separation
  factor, SI,A, which is the change in the ratio of
  interferent to analyte caused by the separation
• In an ideal separation RA 1, RI 0, and SI,A
  0.
• In general, the separation factor should be
  approximately 107 for the quantitative analysis
  of a trace analyte in the presence of a macro
  interferent,
• and 103 when the analyte and interferent are
  present in approximately equal amounts.

9
Example
10
Introduction to Chromatographic Separation
What is chromatography?
11
Historical background of chromatography
Mikhail Tswett invented chromatography in 1906
during his research on plant pigments. He used
the technique to separate various plant pigments
such as chlorophylls, xanthophylls and
carotenoids.
Mikhail Tswett Russian Botanist (1872-1919)
12
Original Chromatography Experiment
End A series of colored bands is seen to form,
corresponding to the different pigments in the
original plant extract. These bands were later
determined to be chlorophylls, xanthophylls and
carotenoids.
Start A glass column is filled with powdered
limestone (CaCO3).
Later
An EtOH extract of leaf pigments is applied to
the top of the column. EtOH is used to flush
the pigments down the column.
13

• Chromatography is a Greek word
• chroma meanscolor and graphein is writing
  color writing
• Tswett named this new technique chromatography
  based on the fact that it separated the
  components of a solution by color

14
Introduction to chromatographic separation
What is Chromatography? Chrom
atography is a technique for separating mixtures
into their components in order to analyze,
identify, purify, and/or quantify the mixture or
components

• Analyze
• Identify
• Purify
• Quantify

Separate
Components
Mixture
15

• The separation of a mixture is by distribution of
  its components between a mobile and stationary
  phase over time
• mobile phase solvent (gas or liquid that
  carries the components)
• stationary phase column packing material (part
  of the apparatus that does not move with the
  sample)
• All methods of chromatography have a stationary
  phase and a moving, or mobile phase
• Chromatography is used by scientists to
• Analyze examine a mixture, its components, and
  their relations to one another
• Identify determine the identity of a mixture
  or components
• Purify separate components in order to isolate
  one of interest for further study
• Quantify determine the amount of a mixture
  and/or the components present in the sample

16
Classification of Chromatographic Methods

• Chromatography can be classified on the basis of
  
• The shape of the solid support
• The nature of the mobile phase
• The mechanism responsible for separation
• Three general categories of chromatography based
  on mobile phase
• liquid chromatography,
• gas chromatography, and
• supercritical-fluid chromatography.
• The mobile phases in the three techniques are
  liquids, gases, and supercritical fluids
  respectively.

17
Classifications based on the physical means by
which the mobile phase and the stationary phase
are brought in to contact ( based on the solid
support material)

• Planar (two dimensional) Chromatography the
  stationary phase is supported on a flat plate or
  in the fibres of a paper.
• Here the mobile phase moves through the
  stationary phase by capillary action or by
  gravity.
• includes
• Paper chromatography
• Thin layer chromatography
  (TLC)
• Characteristics
• Quick, easy, qualitative and
  quantitative
• two dimensional chromatography is operated using
  liquid
• only as a mobile phase.

18

• 2. Column chromatography is a chromatographic
  technique in which the stationary phase is packed
  (coated) in a glass /metal column.
• Characteristics
• Capable of resolving complex mixtures
• Provides quantitative and qualitative analytical
  data
• All other chromatographs are categorized under
  column chromatography

19
N.B Only LC can be performed on planar and
column techniques
20
Classification according to the force of
separation (mechanism of separation)

• 1- Adsorption chromatography
• SP- solid MP- liquid or gaseous
• Solute is adsorbed on the surface of the solid
  particles. The more strongly a solute is
  adsorbed, the slower it travels through the
  column

21
2- Partition chromatography

• A liquid stationary phase is bonded to a solid
  surface, which is typically the inside of the
  silica (SiO2) chromatography column in gas
  chromatography.
• Solute equilibrates between the stationary liquid
  and the mobile phase, which is a flowing gas in
  gas chromatography.

22
3- Ion exchange chromatography

• SP ion exchange resin MB liquid
  containing the sample
• Solute ions of the opposite charge are attracted
  to the stationary phase

23
4- Gel filtration(size exclusion) chromatography

• SP Porous polymeric matrix formed of spongy
  particles, with pores completely filled with the
  liquid mobile phase (gel).
• This technique separates molecules by size, with
  the larger solutes passing through most quickly.

No attractive interaction between the SP and the
solute
24

• 5- Affinity chromatography
• most selective kind of chromatography employs
  specific interactions between one kind of solute
  molecule and a second molecule that is covalently
  attached (immobilized) to the stationary phase.
• SP Support with immobilized ligand

When a mixture containing a thousand proteins is
passed through the column, only the one protein
that reacts with the antibody binds to the
column. After all other solutes have been washed
from the column, the desired protein is dislodged
by changing the pH or ionic strength.
25
How Does Chromatography Work?

• In all chromatographic separations, the sample is
  transported in a mobile Phase
• The mobile phase is then forced through a
  stationary phase (SP) held in a column or on a
  solid surface.
• Therefore, separation of sample in to their
  components is based on the d/ce in the migration
  rates.
• Samples that interact greatly with SP, then
  appear to move more slowly.
• Samples that interact weakly, then appear to move
  more quickly. Consequence separate bands, or
  zones are obtained (use full for qualitative and
  quantitative purpose)

26
Paper Chromatography(PC)

• Principle
• PC is a planar chromatography
• The technique of PC consists of a sheet of
  cellulose filter paper which serves as a
  stationary phase or separation medium
• A small amount of solute is placed in a small
  area near the end of strip
• A solvent is allowed to move from the end of the
  paper by capillary action
• and after equilibration for some fixed period,
  the solute migrates from its initial point of
  application.

27

• The components of mixture are separated
  completely or partially in distinct coloured
  zones
• The separation is also based on polarity
• On PC the cellulose filter paper acts as polar
  since it contains hydroxyl groups and adsorbed
  water molecules
• Therefore the non polar analyte move fast and the
  polar analyte move slow.
• Mobile phase/solvent pure of solvent may be used
  but a mixture of solvents is preferred

28
Interpreting pc chromatogram

• From the out put of the chromatogram
• Identification can be carried out by running
  standards(known substance) on the same plate
  comparing and their Rf values
• Checking of purity can be done by counting
  /observing on the number of spots developed
• The Rf (retardation factor)value of the sample
  is given by
• where, ds and dm are linear distances measured
  from the line of origin

29

• By definition, Rf value cannot exceed 1.0.
• Ideally, Rf values must be in the range of 0.1 to
  0.9 with a minimum separation of 0.05
• The Rf values are influenced by the impurities in
  the paper and solvent, temperature , saturation
  of the atmosphere and development time

30
Criteria may be adopted for the choice of solvent
on PC

• The solvent should not react chemically with any
  the components of the sample
• The composition of solvent mixture should not
  change with time
• The solvent should not interfere with the
  detection of spots
• The distribution ratio should be independent of
  solute concentration

31
Techniques of development with various flow
directions
Ascending development
Radial development
Descending development
Fast development
32

• Example
• In a paper chromatographic separation of cations-
  Ag, Pb and Hg, the solvent front rises to 18.4
  cm while cationic spots were observed at 15.8,
  12.1 and 5.9 cm, respectively. Calculate Rf
  values of the metal ions.
• solution Rf value for Ag 15.8/18.4 0.86
• Rf value for Pb 12.1/18.4
  0.66
• Rf value for Hg 5.9 /18.4
  0.32

33
Thin layer chromatography (TLC)

• Is a planar chromatography in which the SP is
  rigidly fixed on a flat supporting media.
• Is a method used for separating mixtures,
  identifying substances and testing the purity of
  compounds.
• Separations in TLC involve distributing a mixture
  of two or more substances between stationary
  phase and mobile phases

34

• The stationary phase is a thin layer of
  adsorbent (usually silica gel or alumina or
  powdered cellulose) coated on a plate.
• Some of the supporting mediums are calcium foil,
  plastic sheet and glass
• The mobile phase is a developing liquid which
  travels up the stationary phase, carrying the
  samples with it.

35

• Components of the samples will separate on the
  stationary phase according to how much they
  adsorb on the stationary phase versus how much
  they dissolve in the mobile phase.
• Choice of solvents

36
(No Transcript)
37
Identifying the Spots (visualization)

• If the spots can be seen, outline them with a
  pencil.
• If no spots are observed, the most common
  visualization technique is to hold the plate
  under a UV lamp.
• Many organic compounds can be visualized by using
  this technique, and many commercially made plates
  often contain a substance which aids in the
  visualization of compounds.

38
Visualizing Agents
39
Interpreting the Data

• The interpretation of TLC chromatogram is similar
  to PC
• For identification the Rf values the
  known(standard substance ) is compared to those
  of unknown substances
• For checking of the purity, observe on the
  number of spots developed
• i.e. an impure sample will often develop as two
  or more spots, while a pure sample will show only
  one spot

40

• Note Rf values often depend on the temperature
  and the solvent used in the TLC experiment.
• Therefore the most effective way to identify
  unknown compound is to spot known substances
  authentic - next to unknown substances on the
  same plate.
• some of the ways to carry out quantitative
  analysis using TLC
• i) quantitative analysis can be carried out by
  constructing a calibration curves using the area
  of the sample spot and the area of standard with
  known concentration
• ii) scrap, dissolve and use chemicals/physical
  method to determine the quantity

41

• Generally in TLC there are two modes of
  developing
• Ascending and Horizontal
• Typical developing chambers used for (a)
  ascending and (b) horizontal thin layer
  chromatography
• In the later case, the sample is placed on both
  the sides of the plate and developed towards the
  centre

42
Paper vs Thin Layer Chromatography
Paper Chromatography Thin-Layer Chromatography
Takes more time Faster and better separation
Little preparation Detects smaller amounts
Cellulose filter paper is act as SP. Sp. is an adsorbent martial coated on to a glass or metal/plastic sheets
Ascending, descending and circular modes are there Ascending and horizontal modes are there
Choice of SP. is limited A wide range of stationary phases are available
43
General Theory of Column Chromatography

• In Column chromatography stationary phase is held
  in a narrow tube through which the mobile phase
  is forced under pressure or under the effect of
  gravity to pass through
• This section focuses mainly on general theory
  which can be applied to any form of column
  chromatography
• With appropriate modifications, this theory also
  can be applied to planar chromatography.

44

• The sample is introduced at the top of the column
  as a narrow band
• As the sample moves down the column the solutes
  begin to separate, and the individual solute
  bands begin to broaden and develop a Gaussian
  profile
• If the strength of each solutes interaction with
  the stationary phase is sufficiently different,
  then the solutes separate into individual bands

45

• This pic. Shows an example of a simple column
  chromatography

46
Elution on Column Chromatography

• Elution involves washing a species through a
  column by continuous addition of fresh mobile
  phase
• The sample is introduced at the head of a column,
  where upon the components of the sample
  distribute themselves between the two phases.
• Introduction of additional mobile phase (the
  eluent) forces the solvent containing a part of
  the sample move down through the column

47
(No Transcript)
48

• The progress of a chromatographic separation is
  monitored with a suitable detector situated at
  the end of the column.
• A plot of the detectors signal as a function of
  time or volume of eluted mobile phase is known as
  a chromatogram
• and consists of a peak for each of the separated
  solute bands.
• A chromatographic peak may be characterized in
  many ways, two of which are shown below

49

• Retention Time/ Retention volume
• The time it takes after sample injection for the
  analyte peak to reach the detector is called the
  retention time ( tR )
• Or Retention volume The volume of mobile phase
  needed to move a solute from its point of
  injection to the detector (Vr).
• The small peak in the left is for a species that
  is not retained by the column, often the sample
  or the mobile phase will contain unretained
  species.
• The time or volume of mobile phase required to
  elute a non-retained components is called the
  columns void time, tM, or void volume or dead
  time /dead volume.

50

• The time tM for the unretained species to reach
  the detector is called the dead time
• The second important parameter is the
  chromatographic peaks width at the baseline, w.
• Baseline width is measured in units of time or
  volume, depending on whether the retention time
  or retention volume is of interest

51
tM retention time of mobile phase (dead
time) tR retention time of analyte (solute) tS
time spent in stationary phase (adjusted
retention time or tr)
52

• Migration rates of solutes
• The effectiveness of a chromatographic column in
  separating two solutes depends in part upon the
  relative rates at which the two species are
  eluted.
• The average linear rate of solute migration ? is
• ? L/tR
• where, L is the length of the column packing
• The rate of migration of the unretained species
  is the same as the average rate of motion of the
  mobile phase molecules.
• The average linear rate of movement u of the
  molecules of the mobile phase is
• u L/tM Where tM, the dead time.
• These rates are determined by the magnitude of
  the equilibrium constants (distribution constant)

53

• Distribution Constants
• The distribution equilibria involved in
  chromatography involve the transfer of an analyte
  between the mobile and stationary phases.
• Amobile Astationary
• The equilibrium constant K for this reaction is
  called the distribution constant, the partition
  ratio, or the partition coefficient,
• K cS/cM
• where cs is the molar concentration of the
  solute in the stationary phase and cM is its
  molar concentration in the mobile phase.
• K is constant over a wide range of solute
  concentrations.

54

• The Rate of Solute Migration The Retention
  Factor
• The retention factor, or capacity factor (k ),
  is an important parameter to describe the
  migration rates of solutes on columns.
• is measure of how strongly a solute is retained
  by the stationary phase
• For a solute A, the retention factor kA or
  capacity factor is defined as
• kA KAVS/VM where KA is the distribution
  constant for the species A.
• kA (tRA - tM )/tM tR and tM are readily
  obtained from a chromatogram.
• where tr is known as the adjusted retention
  time
• When the retention factor for a solute is lt 1,
  elution occurs so rapidly that accurate
  determination of the retention times is
  difficult.

55

• When the retention factor is larger gt 20, elution
  times become inordinately long.
• Ideally, separations are performed under
  conditions in which the retention factors for the
  solutes in a mixture lie in the range between 1
  and 5.
• Example In a chromatographic analysis of
  low-molecular-weight acids, butyric acid elutes
  with a retention time of 7.63 min. The columns
  void time is 0.31 min. Calculate the capacity
  factor for butyric acid
• Solution

56

• Relative Migration Rates The selectivity
  Factor
• The selectivity factor (separation factor) ? of a
  column for the two species A and B is defined as
• ? KB/KA
• where KB is the distribution constant for the
  more strongly retained species B and KA is the
  distribution constant for species A. ? is always
  greater than unity.
• A relationship between the selectivity factor
  and retention factors
• ? kB/kA
• Where kB and kA are the retention
  factors/capacity factors. An expression for the
  determination of ? from an experimental
  chromatogram

57
Example

• In the same chromatographic analysis for
  low-molecular-weight acids considered in the
  above example the retention time for isobutyric
  acid is 5.98 min. What is the selectivity factor
  for isobutyric acid and butyric acid?
• Solution First we must calculate the capacity
  factor /retention factor for isobutyric acid
• And from the above example
• The selectivity factor, therefore is

58

• Column efficiency
• The plate and rate theories of chromatography
• Plate theory
• A chromatographic column is made up of numerous
  discrete but contiguous narrow layers called
  theoretical plates.
• At each plate, equilibration of the solute
  between the mobile and stationary phase was
  assumed to take place
• Movement of the solute down the column was then
  treated as a stepwise transfer of equilibrated
  mobile phase from one plate to the next.

L
Chromatographic column with N theoretical plates
59

• Plate height (H) height equivalent of a
  theoretical plate (HETP)
• 2. Plate count (N)/plate number
• The two are related by the equation
• N L/H
• where L is the length (usually in centimeters)
  of the column packing
• These two related terms are widely used as
  quantitative measures of chromatographic column
  efficiency
• Columns efficiency improves with an increase in
  the number of theoretical plates or a decrease in
  the height of a theoretical plate.
• Limitation of plate theory doesnt describe
  /explain the effect of variables that affect band
  broadening
• Band broadening The increase in a solutes
  baseline width as it moves from the point of
  injection to the detector.

60
L length of column packing
s ? standard deviation s2/L? variance per unit
length
61
Relation between column distance and retention
times
62
Relation between column distance and retention
times
96 ? 2?
Tangent at Inflection point
63
Determining the Number of Theoretical Plates
W1/2
64
Example

• A chromatographic analysis for the chlorinated
  pesticide Dieldrin gives a peak with a retention
  time of 8.68 min and a baseline width of 0.29
  min. How many theoretical plates are involved in
  this separation? Given that the column used in
  this analysis is 2.0 meters long, what is the
  height of a theoretical plate?
• Solution

65
Peak Capacity

• Is the maximum number of solutes that can be
  resolved on a particular column (nc)
• where Vmin and Vmax are the smallest and largest
  volumes of mobile phase in which a solute can be
  eluted and detected.
• A column with 10,000 theoretical plates, for
  example, can resolve no more than
• i.e. if the minimum and maximum volumes of mobile
  phase in which the solutes can elute are 1 mL and
  30 mL
• This estimate provides an upper bound on the
  number of solutes that might be separated and may
  help to exclude from consideration columns that
  do not have enough theoretical plates to separate
  a complex mixture.

66

• Example In a 25.0 cm long column, the solvent
  took 2.35min. to run through whereas two
  compounds X and Y took 9.87min and 10.63min with
  peak half width 45.6sec. and 53.4 sec.,
  respectively.
• Calculate
• a) Capacity factor for X and Y.
• b) Separation factor .
• c) Average number of plates and plate
  height.
• d) Resolution.

67

• Solution

68
(No Transcript)
69

• Rate theory of chromatography
• Gives an explanation for the factors that affect
  band/ zone broadening
• The magnitude of kinetic effects on column
  efficiency depends upon the length of time the
  mobile phase is in contact with the stationary
  phase, which in turn depends upon the flow rate
  of the mobile phase.
• Efficiency studies have generally been carried
  out by determining H as a function of
  mobile-phase velocity u.

70

• Relationship between Plate Height and Column
  Variables (Effects of 3 factors on H)
• The van Deemter equation can be written in the
  form
• H A B/u Cu
• A B/u (Cs CM)u
• where H is the plate height in centimeters, u is
  the linear velocity of the mobile phase in
  centimeters per second,
• And the quantities A, B, and C are coefficients
  related to the phenomena of multiple flow paths,
  longitudinal diffusion, and mass transfer between
  phases, respectively.

71

• The C coefficient can be broken into two
  coefficients, one related to the stationary phase
  (Cs) and one related to the mobile phase (CM).
• The van Deemter equation contains terms linearly
  and inversely proportional to, as well as
  independent of the mobile phase velocity.

72

• A. The Multipath Term(A) ( Eddy Diffusion)
• Zone broadening arises in part from the multiple
  of pathways
• Solute molecules passing through a
  chromatographic column travel separate paths that
  may differ in length.
• Because of these differences in path length,
  solute molecules injected simultaneously elute at
  different times.
• The principal factor contributing to this
  variation in path length is
• a nonhomogeneous packing of the stationary phase
  in the column and
• differences in particle size and packing
  consistency cause solute molecules to travel
  paths of different length

73
Multiple Pathways

• The Eddy Diffusion is
• Directly proportional to the diameters of packing
• Not significant at low velocities
• where ordinary diffusion
• effectively averages effects of
• eddy diffusion
• In unpacked (capillary)
  columns, A is zero.

• Some solute molecules follow relatively straight
  paths through the column, but others follow a
  longer, more tortuous (full of twists and
  turns)path

74

• A will depend on irregularity of packing and
  diameter of the particles, i.e.

75

• The Longitudinal Diffusion Term (B/u)
• Longitudinal diffusion in column chromatography
  is a band broadening process in which solutes
  diffuse from the concentrated center of a zone to
  the more dilute regions ahead of and behind the
  zone center.
• The contribution of longitudinal diffusion is to
  be inversely proportional to the mobile phase
  velocity.
• B is directly proportional to the solute
  diffusion coefficient in the mobile phase, DM.
• The higher the u, the smaller the H
• Longitudinal diffusion is much smaller in LC than
  in GC

76

• This

77

• Mass-transfer Coefficients (Cs and CM)
• The need for the two mass-transfer coefficients
  Cs and CM arises because the equilibrium between
  the mobile and the stationary phase is
  established so slowly that a chromatographic
  column always operates under nonequilibrium
  conditions.
• The mass-transfer effect on H is directly
  proportional to u because
• the solute residence time is longer at low u, the
  deviation from equilibrium is less, and zone
  broadening or H is smaller
• (Reading assignments about the details of
  mass-transfer coefficients)

78
Longitude vs. Mass Transfer

• Both longitudinal broadening and mass transfer
  broadening depend upon the velocity of the
  carrier gas
• Longitudinal diffusion
• the direction of movement of solute molecules
  tend to be parallel to the flow of the gas(mp)
• Mass Transfer
• Diffusion tends to be right angles to the flow
• The faster the mobile phase moves, the larger the
  band broadening

79
Van Deemter plot and zone broadening

• From the van Deemter plot depicting the effect of
  carrier gas velocity on the factors affecting the
  plate height,
• it is clear that A is not affected, B decreases
  and
• C increases with the carrier gas velocity. The
  combined effect on the
• plate height gives a minimum at a certain value
  of carrier gas velocity. The velocity at which
  HETP (H)min is obtained is known as optimum
  carrier gas velocity (Uopt.).

• This is known as van Deemter plot

80

• Methods for Reducing Zone Broadening
• Two important controllable variables that affect
  column efficiency are the diameter of the
  particles making up the packing and the diameter
  of the column. Therefore
• This the column diameter should be narrow.
• The column should be packed with smaller
  particles. The packing should be compact.
• The thickness of the liquid layer in the column
  should be minimized

81

• With gaseous mobile phases, the rate of
  longitudinal diffusion can be reduced appreciable
  by lowering the temperature and thus the
  diffusion coefficient DM.
• The consequence is significantly smaller plate
  heights at low temperatures
• And for gas mobile phase using high molecular
  weight of carrier gases

82

• Column Resolution
• The resolution Rs of a column is a measure of its
  ability to separate two analytes. Column
  resolution is defines as
• It is evident from the fig. below that a
  resolution of 1.5 gives an essentially complete
  separation of the two components, whereas a
  resolution of 0.75 does not.
• At a resolution of 1.0, zone A contains about 4
  B and zone B contains a similar amount of A.
• At a resolution for 1.5, the overlap is about
  0.3 and is enough for quantitative analysis
• The resolution for a given stationary phase can
  be improved by lengthening the column, thus
  increasing the number of plates.

83
(No Transcript)
84
Example

• In a chromatographic analysis of lemon oil a peak
  for limonene has a retention time of 8.36 min
  with a baseline width of 0.96 min. g-Terpinene
  elutes at 9.54 min, with a baseline width of 0.64
  min. What is the resolution between the two
  peaks?
• Solution

85

• Optimizing Chromatographic Separations
• Relationship between the resolution of a column
  and the retention factors kA and kB for two
  solutes, the selectivity factor , and the number
  of plates
• Since we are only interested in the resolution
  between solutes eluting with similar retention
  times, it is safe to assume that the peak widths
  for the two solutes are approximately the same
• After some rearranging of the previous equations
  the resolution between the chromatographic peaks
  for solutes A and B is given by
• where kB is the retention factor of the
  slower-moving species

86

• The above equation can be rearranged to give the
  number of plates needed to realize a given
  resolution
• The time (tR)B required to elute the two species
  in with a resolution of Rs is given by
• where u is the linear velocity of the mobile
  phase.

87

• To improve resolution
• increase N
• increase ?
• 2 lt k lt 5 is best (for complex mixtures, 1 lt k
  lt 20)
• See the following table to observe their relation
  ship
• Reading Assignments
• Using the Capacity Factor to Optimize Resolution
• Using Column Selectivity to Optimize Resolution
• Using Column Efficiency to Optimize Resolution

88
(No Transcript)
89

• Example
• Substances A and B have retention times of 16.40
  and 17.63 min, respectively, on a 30.0-cm column.
  An unretained species passes through the column
  in 1.30 min. The peak widths (at base) for A and
  B are 1.11 and 1.21 min, respectively.
• Calculate (a) the column resolution, (b) the
  average number of plates in the column, (c) the
  plate height, (d) the length of column required
  to achieve a resolution of 1.5, and (e) the time
  required to elute substance B on the column that
  gives an Rs value of 1.5.

90

• Example a)
• b)

91
(No Transcript)
92

• APPLICATIONS OF CHROMATOGRAPHY
• Chromatography has grown to be the premiere
  method for separating closely related chemical
  species.
• In addition, it can be employed for qualitative
  identification and quantitative determination of
  separated species.

93

• Qualitative Analysis
• A chromatogram provides only a single piece of
  qualitative information about each species in a
  sample, namely, its retention time.
• It is a widely used tool for recognizing the
  presence or absence of components of mixtures
  containing a limited number of possible species
  whose identities are known.
• Positive spectroscopic identification would be
  impossible without a preliminary chromatographic
  separation on a complex sample.

94

• Quantitative Analysis
• Chromatography can provide useful quantitative
  information about the separated species.
• In a quantitative analysis, the height or area of
  an analytes chromatographic peak is used to
  determine its concentration
• Most modern chromatographic instruments are
  equipped with digital electronic integrators that
  permit precise estimation of peak areas.
• If such equipment is not available, manual
  estimate must be made.