In The Name of Allah: COURSE:-626
In The Name of Allah: COURSE:-626
In The Name of Allah: COURSE:-626
Department of Chemistry
Federal Urdu University Arts,
Science
And
Technology
2021
Name: - Noor alam
Teacher: - ma’am shamma
Topic:-chromatography (Gc/lc/ion
exchange chromatography
COURSE:-626
Chromatography
What is it?
Some materials appear homogenous, but are actually a
combination of substances. For example, green plants contain a
mixture of different pigments. In addition, the black ink in the pens
that are used in this experiment is a mixture of different colored
materials. In many instances, we can separate these materials by
dissolving them in an appropriate liquid and allowing them to
move through an absorbent matrix, like paper.
Chromatography is a method used by scientists for separating
organic and inorganic compounds so that they can be analyzed and
studied. By analyzing a compound, a scientist can figure out what
makes up that compound. Chromatography is a great physical
method for observing mixtures and solvents.
The word chromatography means "color writing" which is a way
that a chemist can test liquid mixtures. While studying the coloring
materials in plant life, a Russian botanist invented chromatography
in 1903. His name was M.S. Tswett.
Chromatography is such an important technique that two nobel
prizes have been awarded to chromatographers. Over 60% of
chemical analysis worldwide is currently done with
chromatography or a variation thereon.
Chromatography is used in many different ways. Some people use
chromatography to find out what is in a solid or a liquid. It is also
used to determine what unknown substances are. The Police,
F.B.I., and other detectives use chromatography when trying to
solve a crime. It is also used to determine the presence of cocaine
in urine, alcohol in blood, PCB's in fish, and lead in water.
Chromatography is used by many different people in many
different ways.
Chromatography is based on differential migration. The solutes in
a mobile phase go through a stationary phase. Solutes with a
greater affinity for the mobile phase will spend more time in this
Phase than the solutes that prefer the stationary phase. As the
solutes move through the stationary phase they separate. This is
called chromatographic development.
How it works
In all chromatography there is a mobile phase and a stationary
phase. The stationary phase is the phase that doesn't move and the
mobile phase is the phase that does move. The mobile phase moves
through the stationary phase picking up the compounds to be
tested.As the mobile phase continues to travel through the
stationary phase it takes the compounds with it. At different points
in the stationary phase the different components of the compound
are going to be absorbed and are going to stop moving with the
mobile phase. This is how the results of any chromatography are
gotten, from the point at which the different components of the
compound stop moving and separate from the other components.
1.1 Introduction
3
Figure 1.1. Categories of Chromatography and Their Relationship to Each
Other.
4
In general, each type of chromatography is comprised of two distinct
steps: chromatography (or separation of individual compounds in distinct elution
bands) and identification (detection of each elution band). Gas chromatography
is the process of taking a sample and injecting it into the instrument, turning the
solvent and analytes into gaseous form, and separating the mixture of
compounds into individual peaks (and preferably individual compounds). Liquid
chromatography completes the same process except the separations occur in a
liquid phase. Individual band or peaks exit the column and identification occurs
by a relatively universal detector. One particularly common detector for both gas
and liquid chromatography is mass spectrometry (MS) which transforms each
analyte from a chemically neutral species into a positive cation, usually breaking
various bonds in the process. Detecting the mass of the individual pieces
(referred to as fragments) allows for conclusive identification of the chemical
structure of the analyte. Principles of gas chromatography (GC) will be covered
in Chapter 2, liquid chromatography (LC) in Chapter 3, capillary electrophoresis
(CE) in Chapter 4 and mass spectrometry (MS) in Chapter 5.
5
comparing the sample analysis to that of a known (reference) compound. The
reference is used to identify the unknown compound by matching retention time
(in chromatography) and ion fragmentation pattern (in mass spectrometry). With
today’s computer mass spectral libraries that contain ion fractionation patterns for
numerous chemicals, the analyst has the option of not using a reference
standard. This is especially valuable if a reference compound is not available or
is expensive. In some cases, especially with low analyte concentration, this
approach may only result in a tentative identification.
This book will focus on GC-MS and LC-MS applications from an analytical
chemistry perspective even though many synthetic chemists will also find much
of this information useful for their applications.
6
In the above figure, the minimum time that a non-retained chemical
species will remain in the system is tM. All compounds will reside in the injector,
column, and detector for at least this long. Any affinity for the stationary phase
results in the compound being retained in the column causing it to elute from the
column at a time greater than tM. This is represented by the two larger peaks
that appear to the right in Figure 1.2, with retention times tRA and tRB. Compound
B has more affinity for the stationary phase than compound A because it exited
the column last. A net retention (tR’A and tR’B) time can be calculated by
subtracting the retention time of the mobile phase(tM) from the peaks retention
time (tRA and tRB).
Figure 1.2 also illustrates how peak shape is related to retention time.
The widening of peak B is caused by longitudinal diffusion (diffusion of the
analyte as the peak moves down the length of the column). This relationship is
usually the reason why integration by area, and not height, is utilized. However,
compounds eluting at similar retention times will have near identical peak shapes
and widths.
7
Chromatographic columns adhere by the old adage “like dissolves like” to
achieve the separation of a complex mixture of chemicals. Columns are coated
with a variety of stationary phases or chemical coatings on the column wall in
capillary columns or on the inert column packing in packed columns. When
selecting a column’s stationary phase, it is important to select a phase
possessing similar intermolecular bonding forces to those characteristic of the
analyte. For example, for the separation of a series of alcohols, the stationary
should be able to undergo hydrogen bonding with the alcohols. When attempting
to separate a mixture of non-polar chemicals such as aliphatic or aromatic
hydrocarbons, the column phase should be non-polar (interacting with the
analyte via van der Waals forces). Selection of a similar phase with similar
intermolecular forces will allow more interaction between the separation column
and structurally similar analytes and increase their retention time in the column.
This results in a better separation of structurally similar analytes. Specific
stationary phases for GC and HPLC will be discussed later in Chapter 2 and 3,
respectively.
8
F ( r c2 ) (L/tm )
2
F (dc /2) (L/tm )
dc
F (L/t m )
4
dc
where cross sectional area of column
4
porosity of column packing
(L/tm ) average linear velocity of mobile phase
In the equations above, rc is the internal column radius, dc is the internal column
diameter, L is the total length of the column, tm is the retention time of a non-
retained analyte (one which does not have any interaction with the stationary
phase). Porosity (e) for solid spheres (the ratio of the volume of empty pore
space to total particle volume) ranges from 0.34 to 0.45, for porous materials
ranges from 0.70 to 0.90, and for capillary columns is 1.00. The average linear
velocity is represented by u-bar.
Vm = tM F Eqn 1.1
mL min. mL/min
and is called the dead volume. For a retained solute, we calculate the volume of
mobile phase needed to move the analyte through the system by
VR = tR F Eqn 1.2
mL min mL/min
In actual practice, the analyst does not calculate the volume of the
column, but measures the flow rate and the retention time of non-retained and
retained analytes. When this is done, note that the retention time not only is the
transport time through the detector, but also includes the time spent in the
injector! Therefore
9
The net volume of mobile phase (V’R) required to move a retained analyte
through the system is
where VR is the volume for the retained analyte and VM is the volume for a
nonretained (mobile) analyte.
t’R F = tR F - tM F
Equation 1.4 is important since it gives the net time required to move a
retained analyte through the system (Illustrated in Figure 1.2, above)
where Pi is the gas pressure at the inlet of the column and Po is the gas pressure
at the outlet. The net retention volume (VN) is
The next concept that must be developed is the partition coefficient (K)
which describes the spatial distribution of the analyte molecules between the
mobile and stationary phases. When an analyte enters the column, it immediately
distributes itself between the stationary and mobile phases. To understand this
process, the reader needs to look at an instant in time without any flow of the
mobile phase. In this “snap-shot of time” one can calculate the concentration of
the analyte in each phase. The ratio of these concentrations is called the
equilibrium partition coefficient,
1
0
where Cs is the analyte concentration in the solid phase and CM is the solute
concentration in the mobile phase. If the chromatography system is used over
tr - tm Vs - Vm
k' Eqn 1.9
tm Vm
analyte concentration ranges where the “K” relationship holds true, then this
coefficient governs the distribution of analyte anywhere in the system. For
example, a K equal to 1.00 means that the analyte is equally distributed between
the mobile and stationary phases. The analyte is actually spread over a zone of
the column (discussed later) and the magnitude of K determines the migration
rate (and tR) for each analyte (since K describes the interaction with the
stationary phase).
Now three ways to quantify the net movement of a retained analyte in the column
have been derived, Equations 1.2, 1.4, and 1.7.
Cs Vs K Vs
k’ Eqn 1.8
C m Vm Vm
where VS/VM is sometimes referred to as b, the volumetric phase ratio.
1
1
Stated in more practical terms, k’ is the additional time (or volume) a
analyte band takes to elute as compared to an unretained analyte divided by the
elution time (or volume) of an unretained band, or
rearranged, gives
tr - tm Vs - Vm
k' Eqn 1.9
tm Vm
L
tr tm (1 k') (1 k') Eqn 1.10
u
where is the linear gas velocity and the parameters in Equation 1.10 were
defined earlier. So, the retention time of an analyte is related to the partition ratio
(k’). Optimal k’ values range from ~1 to ~5 in traditional packed column
chromatography, but the analysts can use higher values in capillary column
chromatography.
KB
k'B
Eqn 1.11
KA k'A
where subscripts A and B represent the values for two different analytes and
solute B is more strongly retained. By this definition, is always greater than 1.
Also, if one works through the math, you will note that
The relative retention time, , depends on two conditions: (1) the nature
of the stationary phase, and (2) the column temperature in GC or the solvent
gradient in LC. With respect to these, the analyst should always first try to select
a stationary phase that has significantly different K values for the analytes. If the
compounds still give similar retention times, you can adjust the column
1
2
temperature ramp in GC or the solvent gradient in LC; this is the general elution
problem that will be discussed later.
Appropriate values of should range from 1.05 to 2.0 but capillary column
systems may have greater values.
First, we will start off with an individual peak and develop a concept called
the theoretical plate height, H, which is related to the width of a solute peak at the
detector. Referring to Figure 1.2, one can see that chromatographic peaks are
Gaussian in shape, can be described by
H 2
Eqn 1.13
L
where H is the theoretical plate height (related to the width of a peak as it travels
through the column), is one standard deviation of the bell-shaped peak, and L
is the column length. Equation 1.13 is a basic statistical way of using standard
deviation to mathematically describe a bell-shaped peak. One standard
deviation on each side of the peak contains ~68% of the peak area and it is
useful to define the band broadening in terms of the variance, 2 (the square of
the standard deviation, ). Chromatographers use two standard deviations that
are measured in time units (t) based on the base-line width of the peak, such that
Eqn 1.14
WL
W 4 4 or Eqn 1.15
4tR
10
Substitution of Equation 1.15 into Equation 1.13, yields
LW
2 Eqn 1.16
H
16t 2 R
L tR 2
N 16 Eqn 1.17
H W
We now have the basic set of equations for describing analyte movement
in chromatography but it still needs to be expanded to more practical applications
where two or more analytes are separated. Such an example is illustrated in
Animation 1.3 for a packed column.
t R,B - t
Rs
R,A
Eqn 1.19
W
t R,B - t R,A N
Rs Eqn 1.20
t 4
R,B
Recall from Equation 1.9, that
tR - m
t
k'
tm
k’B - k’A N
Rs Eqn 1.21
1 k’B 4
Recall that we are trying to develop an equation that relates resolution to
respective peak separations and although k’ values do this, it is more useful to
express the equation in terms of , where = k’B/k’A. Substitution of into
Equation 1.21, with rearrangement, yields
-1 k’ N
Rs B
Eqn 1.22
1 k’B 4
or the analyst can determine the number of plates required for a given
separation:
2 1 k’ 2
N 16 R s
2
B
Eqn 1.23
1 k’B
Thus, the number of plates present in a column can be determined by direct
inspection of a chromatogram, where Rs is determined from Equation 1.19, k’A
and k’B are determined using Equation 1.9, and is determined using Equation
1.11.
tR,B - tR,A
Rs Eqn 1.18
W
tr -t
k’ m
Vs - Vm Eqn 1.9
tm Vm
KB k'B
Eqn 1.11
KA k'A
Table 1.1 Integrator Output for the Chromatogram shown in Figure 1.3.
Example 1.1
Calculate k’, , Rs, H, and N for any two adjacent compounds in Table 1.1.
Solution:
Using peaks eluting at 13.724 and 13.359 minutes the following values
were obtained.
tR - t M 13.359 - 1.782
k' = = = 6.50
tM 1.782
t - tM 13.724 - 1.782
= RB = = 1.03
tRA - tM 13.359 - 1.782
L W2 (30 m) (0.090)2 -5
H = = = 8.51 x 10 m or 85.1 m
16 t 2R 16 (13.359)2
L 30 m
N = = = 352,519 plates
H 8.51 x 10-5 m
t - t
R = RB RA N 13.724 - 13.359 352,519 = 4.12
=
t RB 4 13.724 4
Problem 1.1
Figure 1.4 and Table 1.2 contain data from an HPLC analysis of four s-Triazines
(common herbicides). Calculate k’, , Rs, H, and N for any two adjacent
compounds. Compare and contrast the results for the resin packed HPLC
column to those of the capillary column in the GC example given above.
Figure 1.4 HPLC Chromatogram of Four Triazines. The analytical column was
an 10.0 cm C-18 stainless steel column with 2 m resin beads.
Table 1.2 Integrator Output for the HPLC Chromatogram shown in Figure 1.4.
Now we will review and summarize this lengthy derivation and these
complicated concepts.Optimization of the conditions of the chromatography
system (mobile phase flow rate, stationary phase selection, and column
temperature or solvent gradient) are performed to achieve base-line resolution
for the most difficult separation in the entire analysis (two adjacent peaks). This
process results in symmetrically-shaped peaks that the computer can integrate to
obtain a peak (analyte) area or peak height. A series of known reference
standards are used to generate a linear calibration line (correlating peak area or
height to analyte concentration) for each compound. This line, in turn, is used to
estimate the concentration of analyte in unknown samples based on peak area or
height.
Mass Transfer to
f(k) d2f
and from the C su =
Ds
Stationary Phase
Mass Transfer in
f’(k) d2p
the Mobile Phase CMu =
DM
where is the mobile phase linear velocity, Ds and Dm are diffusion coefficients in
the stationary and mobile phases respectively, df and dp are the diameter of the
packing particles and the thickness of liquid coating on the stationary phase
particles respectively, k is the unitless retention or capacity factor, and f(k) and
f’(k) are mathematical functions of k.
If the linear velocity of the mobile phase is too high, the entire “packet” of a
given analyte will not have time to completely transfer between the mobile and
stationary phase or have time to completely move throughout a given phase
(phases are coated on the column walls and therefore have a finite thickness).
This lack of complete equilibrium of the analyte molecules will result in peak
broadening for each peak or skewing of the Gaussian shape. This, in turn, will
increase H and decrease resolution.
The green line in Figure 1.5 represents the van Deemter curve, the
combined result of the three individual phenomena. Since the optimum operating
conditions has the smallest plate height; the flow rate of the GC should be set to
the minimum of the van Deemter curve. For gas chromatography this occurs
around a linear velocity of 15 to 20 cm/s. However, in older systems, as the oven
and column were temperature programmed, the velocity of the gas changed
which in turn changed the mobile phase flow rate and the linear velocity. This
has been overcome in modern systems with mass flow regulators, instead of
pressure regulators, that hold the linear velocity constant.
The next chapters of this book will focus on the components of GC, LC, and MS
with an additional chapter on interpretation of MS fragmentation patterns. Both GC and LC
rely on the chromatography theory discussed in this chapter and all instruments rely on
some form of calibration if quantitative results are required.
This article describes the basics and fundamentals of GC with tips on the
instrumental operations in laboratory use. There may be other more advanced applications
which require GC customization (aka Process GC or System GC), which will not be
covered in this primer
Data of Chromatography
Chromatogram
Basic instrumentation of GC
As shown in Figure 1, the GC consists of a fow control section, a sample injection
port, a column, a column oven, and a detector in which is connected to a data processor.
Carrier gases such as helium (He), nitrogen (N2) or hydrogen (H2) are preferred to be
supplied at a constant fow rate to the sample to the injection port. A separation tube called
a column is connected between the sample injection port and the detector, all three parts
are maintained at an appropriate temperature. The sample injected into the sample
injection port instantaneously vaporizes and fows into the column with the carrier gas. In
the column, a liquid stationary phase (for example, silicone polymers) is chemically
bonded or coated, the vaporized sample is repeatedly dissolved and vaporized in the liquid
stationary phase and travels downstream with the carrier gas.
Since the process of dissolving and vaporizing the sample in the stationary phase
depends on the physicochemical properties such as the boiling point and the nature of the
column, the time of dissolving in the liquid phase and the time of vaporizing will be different
for each compound. Therefore, even when mixed components are injected, the time for
the components to arrive at the column exit is different, and separation can be detected.
The column exit connects to a detector and when substances other than the carrier gas
are eluted from the column, the detector converts them into electrical signals which are
amplifed and sent to a data processor. By analyzing the electric signal of the detector on
the data processor, the GC will be able to identify the sample and determine its quantity.
Under certain conditions, the time to reach the detector (retention time) is the same after
injection of the compound. By injecting the standard sample and the unknown sample, and
comparing the retention times, quantitation can be done by comparing the sizes of their
peaks.
Applications of GC
Industry Type of Analysis
Pharmaceutical Residual solvent analysis
Food and beverages Component analysis, food safety analysis,
halal analysis of alcohol
Environmental Air, water, soil
Petrochemicals Simulated distillation, component analysis
Material, polymer, additive, gas purity
Chemicals analysis, gas emission in automotive
Energy and gas Artificial photosynthesis research
Figure 2 shows the fow diagram of split injection. The sample is introduced into the
inlet and is vaporized, moving downstream. Large portions of the vaporized sample have
to be exhausted before the sample is separated in the column and detected. The split line
has a role to protect the column from any sample overload and increase the moving
velocity of the component in the injector port.
The split ratio indicates “the ratio between the column fow rate and the split fow
rate” and indicates the approximate branching ratio of the injected sample. When the split
ratio is high (for example 1:400), the rate of the injected sample being introduced into the
column decreases as well as the sensitivity. On the other hand, when the split ratio
is low (for example 1:30), the rate of introduction into the column increases as the
sensitivity increases. The commonly used split ratio is 1:50 to 1:100 in the column with the
inner diameter of 0.25 mm to 0.32 mm but considering the required sensitivity and the load
amount on the column, the analysis is performed using the optimum split ratio.
Glass inserts used in the split method are flled with fllers such as glass wool which
have been deactivated. Quantity values and reproducibility may differ if wool quality,
quantity, and the packing position are different. Since most manufacturers provide the
specifc requirements for wool and position, it is better to adhere to this. The split method is
a relatively simple sample injection method. Higher quality data can be obtained by
understanding and analyzing features well.
Sample
injection needle purge
port
Trap gas
49m L/min
Split
Split vent
0mL/min 49mL/min
Cold OCI is suitable for analyzing compounds that are unstable to heat (easy to
decompose). Due to there being no compositional change of samples, it is an analysis
method with high accuracy for measures such as area value reproducibility. Samples with
low concentrations (less than about 200 ppm per component) are also suitable.
Septum
Sample Carrier
3mLgas
/m
9mL/m
Upper
part of
0.53mm
Empty,
inactive
treated 0. 25 - 0.53mm
b. PTV Injection System (Programmable Temperature Vaporizer)
At the point of sample injection, the injection port is set to a low temperature,
typically below the boiling point of the solvent. Thereafter the PVT is rapidly heated to
vaporize the entire sample, along with the solvent. This method is suitable for analyzing
compounds that are unstable to heat (easy to decompose) with little change in
composition due to the warming of the remaining components at the tip of the syringe
needle.
Unlike Cold On-Column Injection analysis, glass inserts can be used in a PTV.
Additionally, both split and splitless modes can be applied onto the PTV. As a result, PTV
can cope with high and low concentration samples. With the right setting, PTV can prevent
nonvolatile contaminants from entering the analytical column altogether. There are two
main types of separation column used in GC available on the market: capillary and packed
columns.
A capillary column typically has 0.1-0.53 mm internal diameter and 10-100 m length
with very high resolution. The material is fused silica and the inside wall is chemically
bonded with the liquid phase. The outside is coated by polyimide resin to increase the
Intensity. The column with 0.25 mm internal diameter and 30 m length is frequently used.
The GC user chooses the column based on certain attributes such as; the target
compounds, the number of components and the instrument configuration. A smaller
diameter and longer column is suitable if higher resolution is needed, while a larger
diameter column can be used if higher resolution is unnecessary
5% Diphenyl
SH-Rtx-5
95% Dimethylpolysiloxane
Low-polar
6% Cyanopropylphenyl
SH-Rtx-1301, 624
94% Dimethylpolysiloxane
4% Cyanopropylphenyl
SH-Rtx-1701
86% Dimethylpolysiloxane
Mid-polar
50% Phenyl
SH-Rtx-17
50% Methylpolysiloxane
SH-Rtx-200 Trifuoropropylmethylpolysiloxane
SH-Rtx-Wax Polyethyleneglycol
High-polar
90% Bis-cyanopropyl
BPX-90
10%
Cyanopropylphenylpolysiloxane
Detector
Example of Example of Minimum
Detector
Detectable Detectable Amount*
Compound
Thermal Conductivity All compounds except for
TCD 10 ppm (10 ng)
Detector carrier gas
Flame Ionization FID Organic compounds 0.1 ppm (0.1 ng)
Detector
Barrier All compounds except for
Univer BID 0.07 ppm (0.07 ng)
Discharge He and Ne
sal
Ionization
Detect
Detector
or
10 ppm (10 ng) in Scan
mode
Mass Spectrometer MS Ionized molecule 0.5 ppm (0.5 ng) in SIM
mode
10 ppb (10 pg) in MRM
mode
Affinity Chromatography
Chromatography is an important biophysical technique that enables the
separation, identification, and purification of the components of a mixture for
qualitative and quantitative analysis.
It is a separation technique in which a mobile phase carrying a mixture is
caused to move in contact with a selectively absorbent stationary phase.
Affinity chromatography is a type of liquid chromatography for the separation,
purification or specific analysis of sample components.
It utilizes the reversible biological interaction or molecular recognition called
affinity which refers to the attracting forced exerted in different degrees
between atoms which cause them to remain in combination.
Example: Enzyme with and inhibitor, antigen with an antibody etc.
It was discovered by Pedro Cuatrecasas and Meir Wilcheck.
References
Skoog, D.A., F.J. Holler, and S.R. Crouch. 2007. Principles of Instrumental
Analysis. 6th Edition. Thomson Publishing USA
Willard, H.W., L.L. Merritt, Jr., J.A. Dean, F.A. Seattle, Jr. 1981. Instrumental
Methods of Analysis, 6th Edition. Wadsworth Publishing Company, Belmont, CA
USA