In The Name of Allah: COURSE:-626

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IN THE NAME OF ALLAH

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.

In paper and thin-layer chromatography the mobile phase is the


solvent. The stationary phase in paper chromatography is the strip
or piece of paper that is placed in the solvent. In thin-layer
chromatography the stationary phase is the thin-layer cell. Both
these kinds of chromatography use capillary action to move the
solvent through the stationary phase.

What is the Retention Factor, Rf ?


The retention factor, Rf, is a quantitative indication of how far a
particular compound travels in a particular solvent. The Rf value is
a good indicator of whether an unknown compound and a known
compound are similar, if not identical. If the Rf value for the
unknown compound is close or the same as the Rf value for the
known compound then the two compounds are most likely similar
or identical.

The retention factor, Rf, is defined as


Rf = distance the solute (D1) moves divided by the distance
traveled by the solvent front (D2)
Rf = D1 / D2
where

D1 = distance that color traveled, measured from center of the band


of color to the point where the food color was applied

D2 = total distance that solvent traveled

The Different Types of Chromatography


There are four main types of chromatography. These are Liquid
Chromatography, Gas Chromatography, Thin-Layer
Chromatography and Paper Chromatography.
Liquid Chromatography is used in the world to test water
samples to look for pollution in lakes and rivers. It is used to
analyze metal ions and organic compounds in solutions. Liquid
chromatography uses liquids which may incorporate hydrophilic,
insoluble molecules.

Gas Chromatography is used in airports to detect bombs and is


used is forensics in many different ways. It is used to analyze
fibers on a persons body and also analyze blood found at a crime
scene. In gas chromatography helium is used to move a gaseous
mixture through a column of absorbent material.

Thin-layer Chromatography uses an absorbent material on flat


glass or plastic plates. This is a simple and rapid method to check
the purity of an organic compound. It is used to detect pesticide or
insecticide residuesin food. Thin-layer chromatography is also
used in forensics to analyze the dye composition of fibers.

Paper Chromatography is one of the most common types of


chromatography. It uses a strip of paper as the stationary phase.
Capillary action is used to pull the solvents up through the paper
and separate the solutes.

The table below summarizes the information from


above.

Type of Applications in Why and What is it


Chromatography the Real World
Liquid test water samples Used to analyze metal
Chromatography to look for ions and organic
pollution, compounds in solutions.
It uses liquids which
may incorporate
hydrophilic, insoluble
molecules.
Gas detect bombs in Used to analyze volatile
2
Chromatography airports, identify gases. Helium is used to
and quantify such move the gaseous
drugs as alcohol, mixture through a
used in forensics to column of absorbent
compare fibers material.
found on a victim
Thin-Layer detecting pesticide Uses an absorbent
Chromatography or insecticide material on flat glass
residues in food, plates. This is a simple
also used in and rapid method to
forensics to analyze check the purity of the
the dye organic compound.
composition of
fibers
Paper separating amino The most common type
Chromatography acids and anions, of chromatography. The
RNA paper is the stationary
fingerprinting, phase. This uses
separating and capillary action to pull
testing histamines, the solutes up through
antibiotics the paper and separate
the solutes.

Introduction, Chromatography Theory, and Instrument Calibration

1.1 Introduction

Analytical chemists have few tools as powerful as chromatography to measure


distinct analytes in complex samples. The power of chromatography comes
from its ability to separate a mixture of compounds, or “analytes”, and
determine their respective identity (chemical structure) and concentration.
Chromatography can be divided into three basic types that include gas, liquid,
and supercritical fluid chromatography. Liquid chromatography can further be
divided into ion exchange, separations based on size, and even extended to
gel- based electrophoretic techniques. This book will provide a basic
introduction to different types of liquid and gas chromatography. The
relationship between each type of chromatography is illustrated in Figure 1.1.

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.

In mass spectrometry, the combination of compound separation and ion


fragment identification (the subject of Chapter 6) yields an extremely powerful
analysis that is said to be confirmatory. Confirmatory analysis means the analyst
is absolutely sure of the identity of the analyte. In contrast, many other individual
techniques and detectors are only suggestive, meaning the analyst thinks they
know the identity of an analyte. This is especially true with most universal GC
and LC detectors since these detectors respond similarly to many compounds.
The only identifying factor in these chromatographic systems is their elution time
from the column. In order to obtain confirmatory analysis the sample would need
to analyzed by at least two or more techniques (for example, different separation
columns) that yield the same results. Mass spectrometry and nuclear magnetic
resonance (NMR) are two confirmatory techniques in chemistry.

At this point, it is important to understand the different applications GC-MS


and LC-MS offer for two different types of chemists, analytical and synthetic
organic chemists. Organic chemists attempt to create a desired chemical
structure by transforming functional groups and intentionally breaking or creating
bonds; in their resulting identification procedures they already have a relatively
good idea of the chemical structure. To characterize the resulting product the
chemist will use Infrared Spectroscopy (IR) to observe functional groups, Mass
Spectrometry (MS) to obtain the compound’s molecular weight, and Nuclear
Magnetic Resonance (NMR) spectroscopy to determine the molecular structure.
Information from all three techniques is used to conclusively identify the
synthesized product.

Analytical chemists are forced to approach identification in a different way,


because they have no a priori knowledge of the chemical structure and because
the analyte is usually present at low concentrations where IR and NMR are
inaccurate. Often, analysis is performed to look for a desired compound by

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.

1.2 Chromatographic Theory

All chromatographic systems have a mobile phase that transports the


analytes through the column and a stationary phase coated onto the column or
on the resin beads in the column. The stationary phase loosely interacts with
each analyte based on its chemical structure, resulting in the separation of each
analyte as a function of time spent in the separation column. The less analytes
interact with the stationary phase, the faster they are transported through the
system. The reverse is true for less mobile analytes that have stronger
interactions. Thus, the many analytes in a sample are identified by retention time
in the system for a given set of conditions. In GC, these conditions include the
gas (mobile phase) pressure, flow rate, linear velocity, and temperature of the
separation column. In HPLC, the mobile phase (liquid) pressure, flow rate, linear
velocity, and the polarity of the mobile phase all affect a compounds’ retention
time. An illustration of retention time is shown in Figure 1.2. The equation at the
top of the figure will be discussed later during our mathematic development of
chromatography theory.

Figure 1.2. Identification of Analytes by Retention Time.

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.

A summary of these concepts and data handling techniques is shown in


Animation 1.1..

Animation 1.1. Baseline Resolution. Go to An_1_1_baselineresolution.mov in


the download folder to view and play.

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.

Derivation of Governing Equations: The development of chromatography


theory is a long established science and almost all instrumental texts give nearly
exactly the same set of symbols, equations, and derivations. The derivation
below follows the same trends that can be found in early texts such as Karger et
al. (1973) and Willard et al. (1981), as well as the most recent text by Skoog et
al. (2007). The reader should keep two points in mind as they read the following
discussion. First, the derived equations establish a relatively simple
mathematical basis for the interactions of an analyte between the mobile phase
(gas or liquid) and the stationary phase (the coating on a column wall or resin
bead). Second, while each equation serves a purpose individually, the relatively
long derivation that follows has the ultimate goal of yielding an equation that
describes a way to optimize the chromatographic conditions in order to yield
maximum separation of a complex mixture of analytes.

To begin, we need to develop several equations relating the movement of


a solute through a system to properties of the column, properties of the solute(s)
of interest, and mobile phase flow rates. These equations will allow us to predict
(1) how long the analyte (the solute) will be in the system (retention time), (2)
how well multiple analytes will be separated, (3) what system parameters can be
changed to enhance separation of similar analytes. The first parameters to be
mathematically defined are flow rate (F) and retention time (tm). Note that “F” has
units of cubic volume per time. Retention behavior reflects the distribution of a
solute between the mobile and stationary phases. We can easily calculate the
volume of stationary phase. In order to calculate the mobile phase flow rate
needed to move a solute through the system we must first calculate the flow rate.

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.

The most common parameter measured or reported in chromatography is


the retention time of particular analytes. For a non-retained analyte, we can use
the retention time (tM) to calculate the volume of mobile phase that was needed
to carry the analyte through the system. This quantity is designated as Vm,

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

where tR is the retention time of the analyte.

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

Vm = Vcolumn + Vinjector + Vdetector

9
The net volume of mobile phase (V’R) required to move a retained analyte
through the system is

V’R = VR - VM Eqn 1.3

where VR is the volume for the retained analyte and VM is the volume for a
nonretained (mobile) analyte.

This can be expanded to

t’R F = tR F - tM F

and dividing by F, yields

t’R = tR - tM Eqn 1.4

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)

Note, for gas chromatography (as opposed to liquid chromatography), the


analyst has to be concerned with the compressibility of the gas (mobile phase),
which is done by using a compressibility factor, j
 P  2 
3 i  1
  Po  
j  
 Pi  2 

2 P  1

 o  


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

VN = j V’R Eqn 1.5

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,

K = CS/CM Eqn 1.6

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).

Equation 1.3 (V’R = VR - VM ) relates the mobile phase volume of a non-


retained analyte to the volume required to move a retained analyte through the
column. K can also be used to describe this difference. As an analyte peak exits
the end of the column, half of the analyte is in the mobile phase and half is in the
stationary phase. Thus, by definition

VRCM = VMCM + VSCS Eqn 1.7

Rearranging and dividing by CM yields

VR = VM + KVS or VR - VM = KVS Eqn 1.8

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.

Now we need to develop the solute partition coefficient ratio, k’ (also


knows as the capacity factor), which relates the equilibrium distribution coefficient
(K) of an analyte within the column to the thermodynamic properties of the
column (and to temperature in GC and mobile phase composition in LC,
discussed later). For the entire column, we calculate the ratio of total analyte
mass in the stationary phase (CSVS) as compared to the total mass in the mobile
phase (CMVM), or

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.

Multiple Analytes: The previous discussions and derivations were


concerned with only one analyte and its migration through a chromatographic
system. Now we need to describe the relative migration rates of analytes in the
column; this is referred to as the selectivity factor, . Notice Figure 1.2 above
had two analytes in the sample and the goal of chromatography is to separate
chemically similar compounds. This is possible when their distribution
coefficients (Ks) are different. We define the selectivity factor as

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

V'B tR,B - tm t'R,B


    Eqn 1.12
V'A t R,A - t m t' R ,A

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.

Now, we finally reach one of our goals of these derivations, an equation


that combines the system conditions to define analyte separation in terms of
column properties such as column efficiency (H) and the number of separation
units (plates, N) in the column (both of these terms will be defined later). As
analyte peaks are transported through a column, an individual molecule will
undergo many thousands of transfers between each phase. As a result, packets
of analytes and the resulting chromatographic peaks will broaden due to physical
processes discussed later. This broadening may interfere with “resolution” (the
complete separation of adjacent peaks) if their K (or k’) values are close (this will
result in an a value close to 1.0). Thus, the analyst needs a way to quantify a
column’s ability to separate these adjacent peaks.

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

Here, L is given in cm and tR in seconds. Note in Figure 1.2, that the


triangulation techniques for determining the base width in time units (t) results in
96% if the area or ±2 standard deviations, or

  
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

H is always given in units of distance and is a measure of the efficiency of


the column and the dispersion of a solute in the column. Thus, the lower the H
value the better the column in terms of separations (one wants the analyte peak
to be as compact as possible with respect to time or distance in the column).
Column efficiency is often stated as the number of theoretical plates in a column
of known length, or

L  tR 2
N   16  Eqn 1.17
H  W 

This concept of H, theoretical plates comes from the petroleum distillation


industry as explained in Animation 1.2 below.

Animation 1.2 Origin of H and the Theoretical Plate Height Unit. Go to


An_1_2_oil.mov in the download folder to view and play.
To summarize Animation 1.2 with respect to gas and liquid
chromatography, a theoretical plate is the distance in a column needed to
achieve baseline separation; the number of theoretical plates is a way of
quantifying how well a column will perform.

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.

Animation 1.3 Separation of Two Analytes by Column Chromatography. Go to


An_1_3_bars.mov in the download folder to view and play.

Separation of two chemically-similar analytes is characterized


mathematically by resolution (Rs), the difference in retention times of these
analytes. This equation, shown earlier in Figure 1.2, is

2(tR' B - t R' A ) Eqn 1.18


R s=
WA + W B
where tR’B is the corrected retention time of peak B, tR’A is the corrected retention
time of peak A, W A is the peak width of peak A in time units and W B is the width
of peak B. Since W A = WB = W, Equation 1.18 reduces to

t R,B - t
Rs 
R,A
Eqn 1.19
W

Equation 1.17 expressed W in terms of N and tR, and substitution of Equation


1.17 into Equation 1.18, yields

 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

substitution into Equation 1.19 and upon rearrangement, yields

 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

Another use of Equation 1.23 is that it can be used to explain improved


separation with temperature programming of the column in GC and gradient
programming in HPLC. Recall that poorer separation will result as peaks
broaden as they stay for extended times in the column and several factors
contribute to this process. N can be changed by changing the length of the
column to increase resolution but this will further increase band broadening. H
can be decreased by altering the mobile phase flow rate, the particle size of the
packing, the mobile phase viscosity (and thus the diffusion coefficients), and the
thickness of the stationary phase film.

To better understand the application of the equations derived above a


useful exercise is to calculate all of the column quantification parameters for a
specific analysis. The chromatogram below (Figure 1.3) was obtained from a
capillary column GC with a flame ionization detector. Separations of
hydrocarbons commonly found in auto petroleum were made on a 30-meter long,
0.52-mm diameter DB-1 capillary column. Table 1.1 contains the output from a
typical integrator.
Figure 1.3 Integrator Output for the Separation of Hydrocarbons by Capillary
Column GC-FID.

Table 1.1 Integrator Output for the Chromatogram shown in Figure 1.3.

Analyte Retention Time Area Peak Width at the


(min) Base (in units of
minutes)
Solvent (tM) 1.782 NA NA
Benzene 4.938 598833 0.099
Iso-octane 6.505 523678 0.122
n-Heptane 6.956 482864 0.100
Toluene 9.256 598289 0.092
Ethyl Benzene 13.359 510009 0.090
o-Xylene 13.724 618229 0.087
m-Xylene 14.662 623621 0.088

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.

Analyte Retention Time Area Peak Width at the


(min) Base (in units of
minutes)
Solvent (tM) 1.301 NA NA
Peak 1 2.328 1753345 0.191
Peak 2 2.922 1521755 0.206
Peak 3 3.679 1505381 0.206
Peak 4 4.559 1476639 0.198

1.3 Optimization of Chromatographic Conditions

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.

An instrument’s resolution can be altered by changing the theoretical plate


height and the number of theoretical plates in a column. The plate height, as
explained in the animation below, is the distance a compound must travel in a
column needed to separation two similar analytes. The number of theoretical
plates in a column is a normalized measure of how well a column will separate
similar analytes.

Now it is necessary to extend the concept of theoretical plate height (H) a


bit further to understand its use in chromatography. Since gas and liquid
chromatography are dynamic systems (mobile flow through the column), it is
necessary to relate a fixed length of the column (the theoretical plate height) to
flow rate in the column. Flow rate is measured in terms of linear velocity, or how
many centimeters a mobile analyte or carrier gas will travel in a given time
(cm/s). The optimization of the relationship between H and linear velocity (),
referred to as a van Deemter plot, is illustrated in Figure 1.5 for gas
chromatography.
Figure 1.5. A Theoretical van Deemter Plot for a Capillary Column showing the
Relationship between Theoretical Plate Height and Linear Velocity.

It is desirable to have the smallest plate height possible, so the maximum


number of plates can be “contained” in a column of a given length. Three factors
contribute to the effective plate height, H, in the separation column. The first is
the longitudinal diffusion, B (represented by the blue line in Figure 1.5) of the
analytes that is directly related to the time an analyte spends in the column.
When the linear velocity () is high, the analyte will only spend a short time in the
column and the resulting plate height will be small. As linear velocity slows, more
longitudinal diffusion will cause more peak broadening resulting in less
resolution. The second factor is the multi-flow path affect represented by the red
line in Figure 1.5. This was a factor in packed columns but has been effectively
eliminated when open tubular columns (capillary columns) became the industry
standard. Third are the limitations of mass transfer between and within the gas
and stationary phases, Cu (the yellow line in Figure 3) defined by

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.

These concepts are reviewed in Animation 1.4.

Animation 1.4 Construction of a van Deemter Curve for an HPLC System. Go to


An_1_4_vandeemtercurve.mov in the download folder to view and play.
Now that the theoretical basis for understanding chromatographic
separation has been established, it is necessary to extend these ideas one step
further. Remember, the power of chromatography is the separation of complex
mixtures of chemicals; not just for two chemicals as illustrated previously. In
most cases separating mixtures of many compounds is required. This requires
that the resolution, Rs, be constantly optimized by maintaining H at its minimum
value in the van Deemter curve.

This optimization is accomplished by systematically altering the column


temperature in GC or the solvent composition in HPLC. Analytes in the
separation column spend their time either “dissolved” in the stationary phase or
vaporized in the mobile phase. When analytes are in the stationary phase they
are not moving through the system and are present in a narrow band in the
length of the column or resin coating. As the oven temperature is increased,
each unique analyte has a point where it enters the mobile phase and starts to
move down the column. In GC, analytes with low boiling points will move down
the column at lower temperatures, exit the system, and be quantified. As the
temperature is slowly increased, more and more analytes (with higher boiling
points) likewise exit the system. In reverse-phase HPLC, analytes with more
polarity will travel fastest and less polar analytes will begin to move as the
polarity of the mobile phase is decreased. Thus, the true power of GC separation
is achieved by increasing the oven/column temperature (referred to as ramping)
while in LC the separation power is in gradient programming (composition of the
mobile phase). This is “the general elution problem” that is solved by optimizing
the mobile phase, linear velocity, and the type of stationary phase. As noted,
temperature programming is used to achieve separation of large numbers of
analytes in GC. An example of the effects of temperature programming on
resolution is illustrated in Figure 1.6. In Figure 1.6a, a low isothermal
temperature is used to separate a mixture of six analytes with limited success as
some peaks contain more than one analyte. A higher isothermal temperature,
shown in Figure 1.6b, is more successful for analytes with higher boiling points
but causes a loss of resolution for peaks that were resolved at the lower
isothermal temperature. The temperature program used to produce Figure 1.6c
achieves adequate separation and good peak shape for a complex solution.
Figure 1.6. Temperature Programming: The Solution to the General Elution
Problem for GC Applications.
1.4 Calibration of an Instrument/Detector

We now have a basis for understanding separation science with respect to


chromatography. All chromatography systems rely on these principles. But how
does the analyst relate instrument output to analyte concentration in a sample?
Instruments yield signals (also referred to as responses) that are specific to the
type of detector being used. Most GC detectors result in electrical currents while
most LC detectors yield absorbance values. MS units can be attached to both
GC and LC systems and yield counts of ions per time. But before actual samples
are analyzed each instrument detector must be calibrated. Two common forms
of calibration are internal and external calibration.

Detector response yields two useful means of quantification in


chromatography: peak area and peak height. In the “old days” these
measurements were made manually; a strip chart recording was obtained by
passing a strip of paper consisting of uniform weight past a pen that moved
relative to the detector signal. The shape of the peak was drawn on the paper
and the peak height was measured with a ruler or the peak area was measured
either by triangulation or by actually weighing a cutout of the paper containing the
peak! Fortunately for us, these archaic methods are no longer required. The
major disadvantage of these techniques is that the range of detector responses
was limited by the height of the paper. Today, peak area and height
measurements are calculated by electronic integrators or computers, and most
systems are automated such that peak area/height are directly correlated
between standards and samples. Most systems use peak area to generate
calibration lines, which are usually linear relationships between the detector
response and the concentration or mass of analyte injected into the instrument.
Such a plot is shown for an external calibration method in Figure 1.7.
Figure 1.7 External Calibration of Benzene on a Capillary Column GC.

A summary of integration concepts is illustrated in Animation 1.5.

Animation 1.5 Integration of Chromatographic Peaks. Go to


An_1_5_shoulderpeaks.mov in the download folder to view and play.

After an instrument has been calibrated, a sample extract is analyzed


under the same conditions as the standards. The calculated area for the sample
is then analyzed by a linear regression of the standard line and a mass or
concentration of the analyte in the sample is calculated. Usually a dilution factor
adjustment is made and the concentration of analyte in the original sample is
then calculated.

A special type of additional calibration is used in capillary column gas


chromatography because of analyte losses during sample injection and due to
the possibility of inconsistent injections when manual injections are preformed.
This method is referred to as an “internal standard” where every sample and
standard injected into the instrument contains an identical concentration of a
compound with similar chemical structure to the analyte but one that has a
unique retention time in the column. The instrument is set to measure a constant
concentration (and therefore measured area) of the internal standard and adjusts
all injections to that constant value. For example, if a sample is found to only
contain 90 percent of the internal standard, then it is assumed that 10 percent of
the injection was lost and all analyte concentrations are increased by 10 percent. Similarly
adjusts can be made of over injecting a sample.

1.5 Evolution of Peak Integration

Calibration curves or lines in chromatography are based on correlating peak area to


analyte concentration. Our ability to more accurately quantify peak area has evolved over
several decades. Starting prior to the 1960s and 1970s with the tracing of chromatographic
peaks on strip charts where the chart paper was then cut out and weighted for correlation
to mass injected, to strip chart integrators, to crude electronic integrators, to stand alone
combination strip chart
– integrator units, and finally to computer based control of the instrument. We have finally
reached the plateau of our quantitation evolution with computers.

The control of our instruments by computers is divided into two programming


components: the sequence and the method. First a method on the computer is created that
controls most instrument functions such as mobile flow rates, temperature zones and
settings, and most of the electronic components of the instrument. The method is directly
linked to the sequence that controls the data collection, including the auto-sampler and
which sample vial is in which slot, what slots in the auto-sampler contain blanks, samples,
and standards, and where the data are stored on the computer for each instrument run.
The combined instrument-computer systems are slightly complicated to learn but greatly
save time, money, and effort in the long run. Most computer controlled systems can also
take a three-to-five day instrument run of 100 samples, create a calibration line, and report
all sample results back to the original sample concentration.

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.

Gas Chromatography (GC) Technique


Gas chromatography (GC) is a powerful tool for the separation of organic molecules
and has been used in several different areas of chemistry research and applications. With
respect to environmental chemistry, GC is routinely used for the analysis of contaminants
and is also used to study the components of complex systems such as gasoline, smoke,
oil, and soil organic matter. The technique essentially involves heating a sample until its
constituent compounds are vapourized into the gas phase, then separating the
components using chromatography and detecting and quantifying the individual
components with a detector and software system.
Chromatography is a general term that refers to the separation of mixtures based
on their affinities for one or more phases
The figure above shows a schematic of the basic components of a gas
chromatograph. The sample injection system was traditionally operated manually but more
modern instruments use automated injection systems that deliver more accurate volumes
with better reproducibility. Newer instruments also have autosamplers that allow for
multiple samples to be analyzed in sequence without the user having to be present. The
injector needle delivers the liquid sample (typically 1-5 μL) to the sample
inlet which is heated to high temperature (~300 °C) which causes the sample to
vapourize. Depending on the sample concentration, the gaseous sample may be diluted
with an inert carrier gas, usually helium but sometimes nitrogen or argon, so that the
sample plug introduced onto the column is not too large. Split injection mode dilutes the
sample with carrier gas by a precise ratio (typically ranging between 1:2 to 1:200) whereas
in splitless mode no sample dilution occurs.
The gas-phase sample is then swept into the capillary column, which is a long,
coiled, thin tube that is approximately 30 metres long but only a fraction of a millimetre in
diameter. The inside of the capillary column is coated in a thin film of material known as
the stationary phase which varies depending on the analyte of interest. A common
stationary phase for nonpolar analytes is composed of silica bonded to long alkyl chains
(C18) that sometimes contains phenyl rings.
The column is housed inside a thermostatted oven whose temperature can be
carefully controlled and increased at different rates as desired. Once the components pass
through the column, they reach a detector which may be a flame ionization detector, mass
spectrometer, or another type of detector which produces a signal that is proportional to
the concentration of each component. The resulting chromatogram is a graph of signal
intensity plotted versus retention time, the time required for the compound to pass through
the column and reach the detector. For quantitative analysis, internal or external standards
of known concentration may be analyzed along with unknown samples. Identification of
molecules can be based on the retention time

of standards or using libraries of standard spectra when a mass spectrometer is


used as the detector.
In order to be analyzed using GC, molecules must be thermally stable at the oven
temperatures used which can sometimes exceed 300 °C. Molecules which thermally
decompose at these temperatures cannot be analyzed by GC. Analyte molecules must
also be volatile and not react with the stationary phase during the chromatographic
separation. To alleviate this, molecules can be derivatized to add functional groups which
improve their volatility and remove reactive functionalities such as hydroxyl groups.
Temperature is a critical experimental parameter in GC. Temperatures that exceed
a compound’s boiling point will increase its volatility and cause it to partition more into the
gas phase. As a result, the compound moves through the column more quickly and
interacts less with the stationary phase. Therefore, carefully controlling the oven
temperature can assist with the separation of similar compounds with small differences in
their boiling points.
As the gas-phase mixture components pass through the column, they are retained
to varying degrees based on their affinity for the stationary phase, thus permitting the
separation of similar compounds. As mentioned previously, a number of different
stationary phase compositions can be used to target specific compounds. For example, a
polar stationary phase consisting of polyethylene glycol units can be used to separate
polar analytes such as free acids whereas a stationary phase with phenyl groups can be
used to target aromatic compounds. All columns have advantages and disadvantages
such as the ability to separate or interact with specific types of compounds as well as the
maximum temperatures that they can withstand before they begin to degrade.
Column bleed refers to the loss of stationary phase material from the column during
a sample run which can contaminate the sample and result in peaks in the chromatogram
which are unexpected and may obscure sample peaks

Gas chromatography (GC


is an analytical methodology, which was
devised by Nobel Laureate, Martin, et al. in 1952. More than 60 years after the award, GC
systems are widely commercialized and used in various industries, capable of both of
quantitation and qualifcation. GC is applicable for many types of analysis in the markets
such as residual solvent analysis in pharmaceuticals, residual pesticides analysis in food
safety, trace level analysis for environmental as well as petrochemical and fne chemical
industry.

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

GC Structure and Fundamentals


Gas chromatograph is an analytical instrument used to analyze the different
components in a sample. An analytical method using a gas chromatograph is called gas
chromatography (GC).
Term Definition

Chromatography Method for Separation

Instrument for Chromatography


Chromatograph

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

Sample Injection Methods


There are many different injection methods: split injection, splitless injection, direct
injection, on-column injection.

Split Injection Method


Split injection is the most popular and versatile method in capillary GC analysis.
Split injection can be applied to many types of analysis, and whilst it may be less sensitive,
the resolution of the chromatogram is not affected.

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

Carrier gas 3m L/min


100 k Pa
50m L/min

Trap gas

49m L/min

Split
Split vent

Sample Septum Sample Septum


injection needle purge injection needle purge

150 k Pa 3mL/min 150 k Pa 3mL/min


2mL/min 50m L/min

5m L/min 53m L/min


1~2 minutes
Vent Trap gas column Vent Trap gas column

0mL/min 49mL/min

Split 2mL/min Split 2mL/min

Splitless Injection Method


The inlet for splitless injection can be shared with split injection. The splitless
injection method is suitable for higher boiling temperature compounds and it is
used to measure lower concentrations in environmental and residual pesticides
feld. Figure 3 shows the conceptual diagram of the splitless injection method. It is
similar to the split injection fow diagram, because when the sampling time is set
to zero, the splitless injector essentially operates like a split injector. The
sampling time is the amount of time set to direct samples into the column, before
the split vent is open.
The operation during sample injection and the fow of carrier gas will be described
with reference to Figure
1. Before injecting the sample, keep the column initial temperature at 50 to 60 °C
and close the split line as shown on the left side of Figure 3. When the sample
is injected into the inlet, it will be vaporized. The vaporized sample gradually
moves to the column due to the carrier gas. At this time, the component with
relatively high boiling point is concentrated in a narrow band at the tip end
portion of the column because the column initial temperature is low. It takes
1 to 2 minutes for most of the vaporized sample to be introduced into the
column, and this is the typical set amount of sampling time in a splitless
injection. Thereafter, as shown in the right diagram of Figure 3, the split line is
opened, the solvent and the sample at the inlet and the column are removed,
the temperature is raised, and the concentrated component is separated at
the tip end portion of the column to detect. with a low analyte concentration.
There are some caveats but it is widely used in environmental and residual
pesticide analysis as a method that can easily achieve high sensitivity.

Direct Injection Method


The majority of the sample is introduced into the capillary column. The inner
diameter of the capillary column is 0.45 mm or more, which is called wide bore. It is
recommended that the column length be 25 m or more. Short columns of 15 m or less are
often diffcult to use due to low set pressure.

Figure 4 shows an example of a structural view of an injection port dedicated to the


wide bore column. In order to suppress sample retention in the glass insert and the wide
bore column connection part, a part of the carrier gas is supplied as a purge gas in the
column connection part, or a taper is formed in the lower part of the insert so that the wide
bore column closely adheres to the bottom part of the insert. When using a septum purge
type total volume injection port, it can be used with a carrier gas fow rate near the optimum
fow rate (about 5 mL /min of He) for a wide bore column.

Cold Injection Method


Cold on-column caps Injection (Cold OCI)
Inlet temperature is kept below the boiling point of the sample solvent and the tip of
the micro syringe is inserted directly into the tip of the capillary column for sample
injection. Thereafter, by raising the temperature of the inlet and column, the sample is
gradually vaporized directly inside the capillary column. The tip of the capillary column
corresponds to the vaporization chamber.

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

Polarity Example of Column Typical Liquid Phase


Squalane
Non-polar
SH-Rtx-1 100% Dimethylpolysiloxane

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

Electron Capture Organic Halogen


ECD 0.01 ppb (0.01 pg)
Detector compounds Organic
mercury compounds
Sulfur compounds
Selecti Flame Photometric Organic phosphorus
FPD 10 ppb (10 pg)
ve Detector compounds
High- Organic tin compounds
sensiti Organic phosphorus
vity Flame 0.1 ppb (0.1
FTD compounds
Detect Thermionic pg) 1 ppb (1
(NPD) Organic nitrogen
or Detector pg)
compounds
Sulfur
Chemiluminesce SCD Sulfur compounds 1 ppb (1 pg)
nce Detector

Ion Exchange Chromatography


 Chromatography is the separation of a mixture of compounds into its
individual components based on their relative interactions with an inert matrix.
 Ion exchange chromatography (or ion chromatography) is a process that
allows the separation of ions and polar molecules based on their affinity to ion
exchangers.
 The principle of separation is thus by reversible exchange of ions between
the target ions present in the sample solution to the ions present on ion
exchangers.
 In this process two types of exchangers i.e., cationic and
anionic exchangers can be used.
 Cationic exchangers possess negatively charged group, and these will
attract positively charged cations. These exchangers are also called “Acidic
ion
 Exchange” materials, because their negative charges result from the
ionization of acidic group.
1. Anionic exchangers have positively charged groups that will attract negatively
charged anions. These are also called “Basic ion exchange” materials.
 Ion exchange chromatography is most often performed in the form of column
chromatography. However, there are also thin-layer chromatographic methods
that work basically based on the principle of ion exchange.
Working Principle of ion exchange chromatography
This form of chromatography relies on the attraction between oppositely charged
Stationary phase, known as an ion exchanger, and analyte.
 The ion exchangers basically contain charged groups covalently linked to the
surface of an insoluble matrix.
 The charged groups of the matrix can be positively or negatively charged.
 When suspended in an aqueous solution, the charged groups of the matrix will
be surrounded by ions of the opposite charge.
 In this “ion cloud”, ions can be reversibly exchanged without changing the
nature and the properties of the matrix.
Instrumentation of ion exchange chromatography
Typical IC instrumentation includes: pump, injector, column, suppressor, detector and
recorder or data system.
1. Pump
The IC pump is considered to be one of the most important components in the system
which has to provide a continuous constant flow of the eluent through the IC injector,
column, and detector.
2. Injector
Sample introduction can be accomplished in various ways. The simplest method is to
use an injection valve. Liquid samples may be injected directly and solid samples need
only to be dissolved in an appropriate solvent. Injectors should provide the possibility
of injecting the liquid sample within the range of 0.1 to 100 ml of volume with high
reproducibility and under high pressure (up to the 4000 psi).
3. Columns
Depending on its ultimate use and area of application, the column material may be
stainless steel, titanium, glass or an inert plastic such as PEEK. The column can vary
in diameter from about 2mm to 5 cm and in length from 3 cm to 50 cm depending on
whether it is to be used for normal analytical purposes, microanalysis, high speed
analyses or preparative work.
Guard column is placed anterior to the separating column. This serves as a protective
factor that prolongs the life and usefulness of the separation column. They are
dependable columns designed to filter or remove particles that clog the separation
column
4. Suppressor
The suppressor reduces the background conductivity of the chemicals used to elute
samples from the ion-exchange column which improves the conductivity
measurement of the ions being tested. IC suppressors are membrane-based devices
which are designed to convert the ionic eluent to water as a means of enhancing the
sensitivity.
5. Detectors
Electrical conductivity detector is commonly use.
6. Data system
In routine analysis, where no automation is needed, a pre-programmed computing
integrator may be sufficient. For higher control levels, a more intelligent device is
necessary, such as a data station or minicomputer.
Procedure of ion exchange chromatography

Ion exchange separations are carried out mainly in columns packed with an
ion-exchanger.
 These ionic exchangers are commercially available. They are made up
of styrene and divinyl benzene. Example. DEAE-cellulose is an anionic
exchanger, CM-cellulose is a cationic exchanger.
 The choice of the exchanger depends upon the charge of particle to be
separated. To separate anions “Anionic exchanger” is used, to separate cations
“Cationic exchanger” is used.
 First the column is filled with ion exchanger then the sample is applied
followed by the buffer. The tris-buffer, pyridine buffer, acetate buffer, citrate
and phosphate buffers are widely used.
 The particles which have high affinity for ion exchanger will come down the
 column along with buffers.
 In next step using corresponding buffer separates the tightly bound particles.
 Then these particles are analyzed spectroscopically.
 An important use of ion-exchange chromatography is in the routine analysis
of amino acid mixtures.
Applications of ion exchange chromatography
 The 20 principal amino acids from blood serum or from the hydrolysis of
proteins are separated and used in clinical diagnosis.
 This is most effective method for water purification. Complete deionization of
water (or) a non-electrolyte solution is performed by exchanging solute cations
for hydrogen ions and solute anions for hydroxyl ions. This is usually achieved
by method is used for softening of drinking water.
 In the analysis of products of hydrolysis of nucleic acids. In this way,
information is gained about the structure of these molecules and how it relates
to their biological function as carriers of hereditary information.
 Chelating resins are used to collect trace metals from seawater.
 To analyze lunar rocks and rare trace elements on Earth.
Advantages of ion exchange chromatography
1. t is one of the most efficient methods for the separation of charged particles.
2. It can be used for almost any kind of charged molecule including large
proteins, small nucleotides and amino acids.
3. Ion exchange is used for both analytical and preparative purposes in the
laboratory, the analytical uses being the more common.
4. Inorganic ions also can be separated by ion-exchange chromatography.

Limitations of ion exchange chromatography



 Only charged molecules can be separated.
 Buffer
 Requirement

Gel permeation chromatography


 Gel permeation chromatography is also called as gel filtration or size exclusion
chromatography.
 In size exclusion chromatography, the stationary phase is a porous matrix made
up of compounds like cross-linked polystyrene, cross-like dextrans,
polyacrylamide gels, agarose gels, etc.
 The separation is based on the analyte molecular sizes since the gel behaves
like a molecular sieve.
 This technique is used for the separation of proteins, polysaccharides, enzymes,
and synthetic polymers.
 As a technique, size exclusion chromatography was first developed in 1955 by
Lathe and Ruthven.

Principle of Gel Permeation Chromatography


 It is a technique in which the separation of components is based on the
difference in molecular weight or size.
 The stationary phase used is a porous polymer matrix whose pores are
completely filled with the solvent to be used as the mobile phase.
 The molecules in the sample are pumped through specialized columns
containing such microporous packing material (gel).
 The basis of the separation is that molecules above a certain size are totally
excluded from the pores, while smaller molecules access the interior of the
pores partly or wholly.
 The flow of the mobile phase hence will cause larger molecules to pass through
the column unhindered, without penetrating the gel matrix, whereas smaller
molecules will be retarded according to their penetration of the gel.
Components/ Instrumentation of Gel Permeation
Chromatography
1. Stationary Phase
2. The Mobile Phase
3. The Columns
4. The Pump
5. Detectors
A. Stationary phase
It is composed of semi-permeable, porous polymer gel beads with a well-defined
range of pore sizes.
It has the following properties:
 Chemically inert
 Mechanically stable
 With ideal and homogeneous porous structure (wide pore size give low
resolution).
 A uniform particle and pore size.
Examples of gel:
1. Dextran (Sephadex) gel: An α 1-6-polymer of glucose natural gel
2. Agarose gel: A 1,3 linked β-D-galactose and 1,4 linked 3,6-anhydro-α, L-
galactose natural gel
3. Acrylamide gel: A polymerized acrylamide, a synthetic gel
B. The Mobile Phase
It is composed of a liquid used to dissolve the bio-molecules to make the mobile phase
permitting high detection response and wet the packing surface.
C. Columns
Any of the following kinds may be used:
 Analytical column- 7.5–8mm diameters.
 Preparative columns-22–25mm
 Usual column lengths-25, 30, 50, and 60 cm.
 Narrow-bore columns- 2–3mm diameter have been introduced
D. Pumps
They are either syringe pumps or reciprocating pumps with a high constant flow rate.
E. Detectors
The detectors may be concentration sensitive detectors, bulk property detectors,
refractive index (RI) detector, etc.
Steps in Gel Permeation Chromatography
It involves three major steps:
A. Preparation of column for gel filtration
It involves:
1. Swelling of the gel
2. Packing the column semi-permeable, porous polymer gel beads with a well-
defined range of pore sizes.
3. Washing: After packing, several column volumes of buffer solution is passed
through the column to remove any air bubbles and to test the column
homogeneity.
B. Loading the sample onto the column using a syringe
C. Eluting the sample and detection of components
Applications of Gel Permeation Chromatography
1. Proteins fractionation
2. Purification
3. Molecular weight determination.
4. Separation of sugar, proteins, peptides, rubbers, and others on the basis of their
size.
5. Can be used to determine the quaternary structure of purified proteins.
 Short analysis time.

Advantages of Gel Permeation Chromatography


 Well defined separation.
 Narrow bands and good sensitivity.
 There is no sample loss.
 The small amount of mobile phase required.
 The flow rate can be set.

Limitations of Gel Permeation Chromatography


 The limited number of peaks that can be resolved within the short time scale of
the GPC run.
 Filtrations must be performed before using the instrument to prevent dust and
other particulates from ruining the columns and interfering with the detectors.
 The molecular masses of most of the chains will be too close for the GPC
separation to show anything more than broad peaks.

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.

Principle of Affinity Chromatography


 The stationary phase consists of a support medium, on which the substrate
(ligand) is bound covalently, in such a way that the reactive groups that are
essential for binding of the target molecule are exposed.
 As the crude mixture of the substances is passed through the chromatography
column, substances with binding site for the immobilized substrate bind to the
stationary phase, while all other substances is eluted in the void volume of the
column.
 Once the other substances are eluted, the bound target molecules can be eluted
by methods such as including a competing ligand in the mobile phase or
changing the pH, ionic strength or polarity conditions.
Components of Affinity Chromatography
1. Matrix
 The matrix is an inert support to which a ligand can be directly or indirectly
coupled.
 In order to for the matrix to be effective it must have certain characters:
 Matrix should be chemically and physically inert.
 It must be insoluble in solvents and buffers employed in the process
 It must be chemically and mechanically stable.
 It must be easily coupled to a ligand or spacer arm onto which the ligand can be
attached.
 It must exhibit good flow properties and have a relatively large surface area for
attachment.
 The most useful matrix materials are agarose and polyacrylamide.
2. Spacer arm
 It is used to improve binding between ligand and target molecule by
overcoming any effects of steric hindrance.
3. Ligand
 It refers to the molecule that binds reversibly to a specific target molecule.
 The ligand can be selected only after the nature of the macromolecule to be
isolated is known.
 When a hormone receptor protein is to be purified by affinity chromatography,
the hormone itself is an ideal candidate for the ligand.
 For antibody isolation, an antigen or hapten may be used as ligand.
 If an enzyme is to be purified,a substrate analog, inhibitor, cofactor, or effector
may be used as a the immobilized ligand.
Steps in Affinity Chromatography
 Affinity medium is equilibrated in binding buffer.
 Sample is applied under conditions that favor specific binding of the target
molecule(s) to a complementary binding substance (the ligand). Target
substances bind specifically, but reversibly, to the ligand and unbound material
washes through the column.
 Elution is performed specifically, using a competitive ligand, or non-
specifically, by changing the pH, ionic strength or polarity. Target protein is
collected in a purified, concentrated form.
 Affinity medium is re-equilibrated with binding buffer.
These events can be summarized into the following three major steps:
1. Preparation of Column
 The column is loaded with solid support such as sepharose, agarose, cellulose
etc.
 Ligand is selected according to the desired isolate.
 Spacer arm is attached between the ligand and solid support.
2. Loading of Sample
 Solution containing a mixture of substances is poured into the elution column
and allowed to run at a controlled rate.
3. Elution of Ligand-Molecule Complex
 Target substance is recovered by changing conditions to favor elution of the
bound molecules.
Applications of Affinity Chromatography
 Affinity chromatography is one of the most useful methods for the separation
And purification of specific products.
 It is essentially a sample purification technique, used primarily for biological
molecules such as proteins.
Its major application includes:
 Separation of mixture of compounds.
 Removal of impurities or in purification process.
 In enzyme assays
 Detection of substrates
 Investigation of binding sites of enzymes
 In in vitro antigen-antibody reactions

Advantages of Affinity Chromatography


 Detection of Single Nucleotide polymorphisms and mutations in nucleic acids
 High specificity
 Target molecules can be obtained in a highly pure state
 Single step purification
 The matrix can be reused rapidly.
 The matrix is a solid, can be easily washed and dried.
 Give purified product with high yield.
 Affinity chromatography can also be used to remove specific contaminants,
such as proteases.
Limitations of Affinity Chromatography
 Time consuming method.
 More amounts of solvents are required which may be expensive.
 Intense labour
 Non-specific adsorption cannot be totally eliminated, it can only be
minimized.
 Limited availability and high cost of immobilized ligands.
 Proteins get denatured if required pH is not adjusted.

References

Karger, B.L., L.R, Synder, C. Horvath. 1973. An Introduction to Separation


Science. 1st Edition, John Wiley and Sons, New York, USA

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

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