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![Page 1: What is chromatography? Chromatography is a powerful separation method that is usually composed of mobile phase and a stationary phase. This method.](https://reader030.fdocuments.in/reader030/viewer/2022033020/56649d205503460f949f52fa/html5/thumbnails/1.jpg)
INTRODUCTION TO CHROMATOGRAPHIC SEPARATIONS
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What is chromatography?
Chromatography is a powerful separation method that is usually composed of mobile phase and a stationary phase.
This method is used to separate and identify the components of complex mixtures.
Works by allowing the molecules present in the mixture to distribute themselves between a stationary and a mobile phase to varying degrees.
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Those components that are strongly retained by the stationary phase move slowly with the flow of mobile phase.
In contrast, components that are weakly held by the stationary phase travel rapidly.
As a consequence of these differences in mobility, sample components separate into discrete bands that can be analyzed qualitatively and/or quantitatively.
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Classification of Chromatographic Methods
Can be categorized based on the followings:
1. Based on physical means The way stationary and mobile phase are
brought into contact
2. Based on the types of mobile phase
Either gas, liquid or supercritical fluid
3. Based on the kinds of equilibria involved in the solute transfer between the phases.
Interaction of analyte between stationary and mobile phases
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Classification of Chromatographic Methods
Stationary phases is held in narrow tube;
mobile phase moves by pressure or gravity
E.g. – gas chromatography (GC)
– supercritical-fluid chromatography (SFC)
Stationary phase is supported on a flat plate or in the interstices of a paper; mobile phase moves through capillary action or gravity
E.g. – thin-layer chromatography (TLC)
– paper chromatography (PC)
Column chromatography
Planar chromatography
* Based upon physical means
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Column chromatography can be further differentiated based on the types of mobile phases and the kinds of equilibria involved in solute transfer between the phases
Mobile Phase
i) Gas
Gas Chromatography
ii) Liquid
Liquid Chromatography
iii) Supercritical fluid
Supercritical-fluid Chromatography
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Types of Chromatography on The Basis of interaction of The Analyte with Stationary Phase
Adsorption – for polar non-ionic compounds Ion Exchange – for ionic compounds
Anion – analyte is anion; bonded phase has positive charge
Cation – analyte is cation; bonded phase has negative charge
Partition – based on the relative solubility of analyte in mobile and stationary phases Normal – stationary phase polar, the mobile phase nonpolar Reverse – stationary phase nonpolar, the mobile phase polar
• Size Exclusion – stationary phase is a porous matrix sieving
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Classification of Chromatographic Methods
Chromatography
Partition
Adsorption
Ion-exchange
Size-exclusion
Liquid-liquid
Gas-liquid
Liquid-solid
Gas-solid
Liquid-solid
Liquid-solid
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PARTITION CHROMATOGRAPHY
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Partition chromatography Accomplished by selective & continuous
transfer of the components of the mixture back & forth between a liquid stationary phase and a liquid mobile phase as the mobile phase liquid passes through the stationary phase liquid
Stationary phase: liquidMobile phase: liquid or gas
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Partitioning distribution (by dissolving) of the
components between 2 immiscible phases: Relative solubilities of the components in the
mobile and stationary phase
e.g. stationary phase – polar Polar components will retain longer than the
non-polar components. Non-polar components will move quickly
through stationary phase & will elute first before the polar components, and vice-versa.
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Partition chromatography
The stationary phase actually consists of a thin film adsorbed (stuck) on or chemically bonded to the surface of a finely divided solid particles.
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Partition chromatography
If the mobile phase is gas, the volatility (vapor pressure) and solubility in stationary phase plays an important role.
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ADSORPTION CHROMATOGRAPHY
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Adsorption (Affinity) Chromatography Components of the mixture selectively
adsorb (stick) on the surface of a finely divided solid stationary phase.
As mobile phase (gas/liquid) carries the mixture through the stationary phase, the components of the mixture stick to the surface of it with varying degrees of strength & thus separate
Stationary phase: solid Mobile phase: gas or liquid
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ION-EXCHANGE CHROMATOGRAPHY
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Ion-exchange chromatography
Method for separating mixture of ions Sample: aqueous solution of inorganic
ions / organic ions Stationary phase – small polymer resin
“beads” usually packed in a glass tube These beads have ionic bonding sites on
their surfaces which selectively exchange ions with certain mobile phase compositions as the mobile phase penetrates through it.
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Ion-exchange chromatography
Ions that bond to the charged site on the resin bead are separated from ions that do not repeated changing of the mobile phase composition.
The usual procedure is to initially use a mobile phase with all the ions in the mixture bond & then to change the mobile phase in a stepwise fashion so that one kind of ion at a time is removed
Done until complete separation achieved
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SIZE-EXCLUSION CHROMATOGRAPHY
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Size-exclusion chromatography
Also called gel permeation chromatography
Technique for separating dissolved species on the basis of their size
Stationary phase: porous polymer resin particles (molecular sieves)
The components to be separated enter the pores of these particles & are slowed from progressing through this stationary phase.
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Size-exclusion chroamtography
Separation depends on the sizes of the pores relative to the sizes of the molecules to be separated
Small particles are retarded to a greater extent than large particles (some of which may not enter the pores at all) & separation occurs.
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TERMINOLOGIES IN CHROMATOGRAPHY
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Terminologies in chromatography
Elution: a process in which species are washed through a chromatographic column by addition of fresh solvent
Mobile phase: is one that moves over or through an immobilized phase that is fixed in place in a column or on the surface of flat plate
Stationary phase: a solid or liquid that is fixed in place. A mobile phase then passes over or through the stationary phase
Retention time: is the time interval btw its injection onto a column and the appearance of its peak at the other end of the column
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Migration Rates of Solutes
Distribution constant, K
Retention time, tR
Capacity factor,k’
Selectivity factor,
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Distribution constant, K
In chromatography, the distribution equilibrium of analytes between the mobile and stationary phases can often be described quite simple.
Let say, we have analyte A. The distribution equilibrium is written as:
A mobile A stationary Therefore, the equilibrium constant K is called
distribution constant and is defined as:
K =
c – Molar concentration
cstationary
cmobileK is also called partition coefficient or partition ratio
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Retention Time, tR
Time required for the sample to travel from the injection part through the column to the detector.
A typical chromatogram for a two-component mixture. The small peak on the left represents a species that is not retained on the column & so reaches the detector almost immediately after elution is started.
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tM - time taken for the unretained species to reach the detector.- sometimes called dead time- Rate of migration of the unretained species is SAME as
the average rate of motion of mobile phase molecules.- So, tM can be expressed as the time required for an average molecule of the mobile phase to
pass through the column.
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Retention Factor (Capacity factor), k’
term used to measure the migration rates of analytes on columns.
k’A = KA (VS / VM) [unitless] for
analyte A
How is k’A related to tR and tM?
k’A =
tR – tM tM
When k’A is 1.0, separation is poorWhen k’A is > 30, separation is slowWhen k’A is 2-10, separation is optimum
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Selectivity Factor,
is defined as: =
=
=
A measure of the relative migration rates of species A and B with a stationary phase material in chromatography
KB
KA
distribution constants
k’B
k’A
capacity factors
tR(B) – tM tR(A) – tM
retention times
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tM
tR
tR
Response
Retention time , min
1 3 6
tR – tM tM
k’ =tR(B) – tM tR(A) – tM
=
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Column Efficiency
Two related terms widely used as quantitative measures of chromatographic column efficiency:
i) Plate height, Hii) Number of theoretical plates, N
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The relationship between H and N is:
N =
The efficiency of chromatographic columns increases as the number of plates becomes greater and plate height become smaller.
LH
Number oftheoretical plates
Column length
Plate height
Efficient column has small plate height
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Experimentally, H and N can be approximated from the width of the base of the chromatographic peak.
The equation:
N = 16
tR
W
2
N can be calculated using tR and W To obtain H, the length of the column must be known
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Another method for approximating N is to determine W½, the width of the peak at half its maximum height.
N = 5.54 tR
2
W½
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Resolution, Rs
A measure of the separation of two chromatographic peaks.
Baseline resolution is achieved when Rs = 1.5
Rs = 2[tR(B) – tR(A)] WA + WB
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Effect of Capacity Factor & Selectivity Factor on Resolution
Relationship btw the resolution of a column and the capacity factor k’, selectivity factor and the number of plates N is given by this equation:
Rs = √N - 1 k’ 4 1 + k’
Simplified: Rs = √N
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Effect Resolution on Retention Time
Relationship btw the resolution of a column and retention time:
tR = 16Rs2H ( 1 + k’)3
u - 1 (k’)2
2
Simplified: tR = Rs
2
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Example
Length of column: 30 cm Peak widths (at base) for A & B were
1.11 & 1.21 min respectively. Calculate:
i) column resolution, Rsii) the average number of plates,
Niii) the plate height, Hiv) length of column to achieve
Rs 1.5
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tM
tR
tR
Response
Retention time , min
1 3 6
1.30 min
16.40 min
17.63 min
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i)
Rs = 2(17.63 min – 16.40 min)
(1.11 min + 1.21 min) = 1.06
ii)
N = 16 16.40 min 1.11 min
= 3.49 x 103
Rs = 2[tR(B) – tR(A)] WA + WB
N = 16
tR
W
2
2
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N = 16 17.63 min 1.21 min
= 3.40 x 103
iii) H = L / N
= 30 cm / 3.44 x 103 = 8.7 x 10-3 cm
iv) (Rs)1 √N1
(Rs)2 √N2
2Therefore, calculatethe N average
Nave = 3.44 x 103
=
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1.06 = √ 3.44 x 103
1.5 √ N 2
N2 = 6.9 x 103
L = N x H = 6.9 x 103 x 8.7 x 10-3
= 60 cm
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BAND BROADENING
Band broadening reflects a loss of column efficiency.
The slower the rate of mass-transfer processes occuring while a solute migrates through a column, the broader the band at the column exit.
Some of the variables that affect mass-transfer rates are controllable and can be exploited to improve separations.
Table 26.2 lists the variables that influence the column efficiency.
Their effect on column efficiency, as measured by the plate height will be described in the following slides
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VARIABLES AFFECTING COLUMN EFFICIENCY
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VARIABLES AFFECTING COLUMN EFFICIENCY
Mobile phase flow rate Particle size Diameter of column Film thickness
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EFFECT OF MOBILE PHASE FLOW RATE ON PLATE HEIGHT
From both the plots for LC and GC, we can see that both show a minimum in H at low linear flow rates.
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EFFECT OF PARTICLE SIZE ON PLATE HEIGHT
Refer to figure 26-11 , page 773 The numbers to the right is the
particle diameters The smaller the particle size, the
more uniform the column packing, then the more tolerant to the change in mobile-phase velocity.
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For packed column, the most important variables that affect column efficiency is the diameter of the particles that making up the packing.
While for open tubular column, the diameter of the column itself is an important variables.
Refer to table 26-3, the mobile phase mass-transfer coefficient CM is known to be inversely proportional to the diffusion coefficient of the analyte in the mobile phase DM.
EFFECT OF DIAMETER OF THE COLUMN ON PLATE HEIGHT
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CM is proportional to the square of the particle diameter of the packing material, d1p (packed column).
CM is proportional to the square of the column diameter, d2p (open tubular column).
As a conclusion, the bigger the column diameter, the smaller the diffusion coefficient DM. therefore, we can say that increase in column diameter will increase the plate height.
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When stationary phase is an immobilized liquid, the mass-transfer coefficient Cs is directly proportional to the square of the thickness of the film on the support particles d1l and inversely proportional to the diffusion coefficient Ds of the solute in the film.
With thick films and smaller diffusion coefficient, analyte molecule travel slower. As a result, slower rate of mass-transfer and an increase in plate height.
EFFECT OF FILM THICKNESS ON PLATE HEIGHT
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Application of Chromatography Qualitative analysis Quantitative analysis
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Qualitative Analysis
Based on retention time
Provided the sample produce the peak at the same retention time as a standard under identical conditions
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Quantitative analysis
Analysis based on Peak Height The height of chromatographic peak is obtained
by connecting the base lines on either side of the peak by a straight line and measuring the perpendicular distance from this line to the peak.
Analysis based on Peak Area Peak areas are usually the preferred method of
quantitation since peak areas are independent of broadening effects.
Most modern chromatographic instruments are equipped with computer or digital electronic integrator that permit precise estimation of peak areas.
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Calibration Method(also known as external method)
- Involve preparation of series of standard solutions that approximate the
composition of the unknown.- The peak heights or areas are plotted as a function of concentration.- The concentration of the component(s) to be analysed is determined by comparing the response(s) peak(s) obtained with the
standard solutions.
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Internal Standard Method
- Equal amounts of an internal standard substance is introduced into each standard and sample.- The internal standard should not react with the substance to be examined; it must be stable
and must not contain impurities with a retention time similar to that of the substance to be examined.- The concentration of the substance to be examined is determined by comparing the ratio
of the peak areas (or heights) due to the substance to be examined and the internal standard in the test solution with the ratio of the
peak areas (or heights) due to the substance to be examined and the internal standard in the
standard solution.
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Area Normalization Method
In the normalization method, the areas of all eluted peaks
The percentage content of one or more components of the substance to be examined is calculated by determining the area of the peak(s) as a percentage of the total area of all the peaks, excluding those due to solvents or any added reagents and those below the disregard limit.
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TAILING AND FRONTING OF CHROMATOGRAPHIC PEAKS
A common cause of tailing and fronting is a distribution constant that varies with concentration.
Fronting also arises when the amount of sample introduced onto a column is too large.
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TYPES OF COLUMN
There are two types of column:
Packed Column Capillary Column
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PACKED COLUMN
Packed column Modern packed columns are fabricated
from glass or metal tubing. These tubes are densely packed with
uniform, finely divided packing material or solid support, coated with a thin layer (0.05 to 1 µm) of the stationary liquid phase.
The tubes are usually formed as coils.
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CAPILLARY COLUMN Capillary column
Also known as open tubular column
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Early wall –coated open tubular (WCOT) column were constructed of stainless steel, aluminium, copper or plastic.later, they are made from glass.
The most widely used capillary columns are fused-silica wall-coated (FSWC) open tubular columns.• Column constructed of fused silica tubing.• Polyimide coating gives it strength (outer layer). This
resulting columns are quite flexible and can be bent into coils.
• These capillaries have much thinner walls compared to glass columns.
• Liquid stationary phases coated or chemically bonded.
Most applications utilize FSWC open tubular column (replacing WCOT glass column).
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Recently, 530-µm capillaries, sometimes called megabore column have appeared on the market.• These columns maintain the features of
capillary column.• These columns tolerate sample sizes that are
similar to those for packed column.• However, the resolution with these columns is
lower compared to capillary column (resolution is higher with column of smaller inner diameter).
• Advantage of megabore column over packed column is their lack of bleeding (loss of stationary phase with time).