Instrumental Technique for Environmental Analysis Chapter 5 Chromatography

Rezaul Karim Environmental Science and Technology Jessore University of Science and Technology

Chapter content

Historical Background; 1,2 Chromatography: A Separation Technique;

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Classification of Chromatography 3 Basic Chromatography Concepts: 2 Chromatogram; Distribution Coefficient; Retention Volume, Capacity Factor, Theoretical Plates, Selectivity Physical Forces and Interactions;2 Modes of Separation; 2 Stationary Phases Versus Mobile Phases; 2 Detectors Applications of Chromatography 3
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Reference
1. Francis Rouessac and Annick Rouessac, 2007, Chemical Analysis-Modern Instrumentation Methods and Techniques, 2nd Edition, John Wiley & Sons Ltd, The Atrium, Southern Gate, England 2. J.R.J. Pare and J.M.R. Belanger 1997 instrumental methods in food analysis, techniques and instrumentation in analytical chemistry- volume 18, Elsevier science . Amsterdam, the Netherlands 3. Skoog, Holler & Crouch 2007, Instrumental Analysis, Brooks Cole Cengage Learning, USA.
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Introduction

Chromatography is a powerful separation method that finds applications in all branches of science,. Chromatography was invented, the Russian botanist Mikhail Tswett. Various plant pigments such as chlorophylls and xanthophylls were separated by passing solutions of these compounds through a glass column packed with finely divided calcium carbonate.
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The separated species appeared as colored bands on the column. the name for the method (Greek chroma meaning "color and graphein meaning "writing"). The tremendous impact in 1952 - Nobel Prize in Chemistry awarded to A. J P, Martin and R. LM, Synge for their discoveries in the field,. Many of the Nobel Prizes awarded since that time have been based on work in which chromatography played a vital role.
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Chromatography as extraction
Chromatography operates on the same principle as extraction, but one phase is held in place while the other moves past it. In the Figure, a solution containing solutes A and B placed on top of a column are packed with solid particles and filled with solvent.

The idea behind chromatography: solute A, with a greater affinity than solute B for the stationary phase, remains on the column longer.
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Fluid entering the column is called eluent. Fluid emerging from the end of the column is called eluate The mobile phase

the solvent moving through the column is either a liquid or a gas.

The stationary phase

the one that stays in place inside the column is most commonly a viscous liquid chemically bonded to the inside of a capillary tube or onto the surface of solid particles packed in the column.
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When the outlet is opened, solutes A and B flow down into the column. Fresh solvent is then applied to the top of the column and the mixture is washed down the column by continuous solvent flow. If solute A is more strongly adsorbed than solute B on the solid particles, then solute A spends a smaller fraction of the time free in solution. Solute A moves down the column more slowly than solute B and emerges at the bottom after solute B. We have just separated a mixture into its components by chromatography.

Theory
Chromatography is the process by which the components of a mixture can be separated. Chromatography is a physico-chemical method of separation of components within mixtures, liquid or gaseous, in the same vein as distillation, crystallization, or the fractionated extraction. It has become one of the primary analytical methods for the identification and quantification of compounds in the gaseous or liquid state.

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The basic principle is based on the concentration equilibrium of the components of interest, between two immiscible phases.
One is called the stationary phase, because it is immobilized within a column or fixed upon a support. The second, called the mobile phase, is forced through the first.

The phases are chosen such that components of the sample have differing solubilities in each phase. The differential migrations of compounds lead to their separation.

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Basic chromatographic process

A basic experiment in chromatography. (a) The necessary ingredients (C, column; SP, stationary phase; MP, mobile phase; and S, sample); (b) introduction of the sample; (c) start of elution; (d) recovery of the products following separation
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A vertical hollow glass tube (the column) is filled with a suitable finely powdered solid, the stationary phase. At the top of this column is placed a small volume of the sample mixture to be separated into individual components. The sample is then taken up by continuous addition of the mobile phase. The mobile phase goes through the column by gravity, carrying the various constituents of the mixture along with it. This process is called elution.
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Elution chromatography
If the components migrate at different velocities, they will become separated from each other and can be recovered, mixed with the mobile phase Each component of a mixture will be associated with a specific retention time. This type of chromatography is termed elution chromatography. Columns are either packed or open tubular.

A packed column is filled with particles of stationary phase. An open tubular column is a narrow, hollow capillary with stationary phase coated on the inside walls.
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Classification of Chromatographic Methods

The first classification is based on the physical means by which the stationary and mobile phases are brought into contact.
In column chromatography, the stationary phase is held in a narrow tube through which the mobile phase is forced under pressure. In planar chromatography, the stationary phase is supported on a flat plate or in the interstices of paper; here, the mobile phase moves through the stationary phase by capillary action or under the influence of gravity.
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one based on the types of mobile and stationary phases and the kinds of equilibria involved in the transfer of solutes between phases. Three general categories:

gas chromatography (GC), liquid chromatography (LC), and Supercritical fluid chromatography (SFC).

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The sample components and the phases

In adsorption chromatography, the sample is attracted to the surface of the phase, usually to the surface of a solid stationary phase. In absorption chromatography / partition chromatography, the sample diffuses into the interior of the stationary phase. Most chromatographic separations are a combination of both adsorption and absorption phenomena.

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Mobile phase indicates the name

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the difference between "gram & "-graph"??

gram = a record -graph = an instrument for recording Examples:

i.

ii.

A telegraph is the piece of equipment used to send the message, sometimes known as a telegram. An electrocardiograph PRODUCES an electrocardioogram. The e'graph records electrical voltage of the heart.

Analogy: chromatograph ,,,,,,,,,,,,,,,,,,chromatogram

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The chromatogram
The chromatogram is the representation of the variation, with time (rarely volume), of the amount of the analyte in the mobile phase exiting the chromatographic column. It is a curve that has a baseline which corresponds to the trace obtained in the absence of a compound being eluted.

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Retention time

Retention time tR

It represents the time elapsed from the sample introduction to the detection of the peak maximum on the chromatogram. A constituent is characterized by its tR In an ideal case, tR is independent of the quantity injected.
formerly designated t0 It is the time required for the mobile phase to pass through the column.

Hold-up time or dead time tM,

The difference between the retention time and the hold-up time is designated by the adjusted retention time of the compound, tR.
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Distribution Coefficient
Also known Nernst partition coefficient It corresponds to the distribution of the analyte (X) between the stationary (s) and mobile (m) phases, as it elutes through the column. Kx = [X]s/[X]m = CS /CM = Molar conc. of the solute in the stationary phase / Molar concentration of the solute in the mobile phase

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Retention volume

The total volume of the mobile phase (Vr) required to elute an analyte is the sum of the volume of the mobile phase (Vm) and the volume of the mobile phase which flows while the analyte is held immobile (Vs*Kx).
Vr = Vm + Vs*Kx

the value of Vr = F*tr , where

F is the flow rate of the mobile phase (volume/time) and the retention time (tr) is the time required for an analyte to elute completely through the column (from injector to detector).
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The dead volume, Vm=F*to

Capacity factor
Capacity factor or Retention factor, kx It is defined by the total number of moles of x in the stationary phase over the total number of moles of x in the mobile phase.

kx = mS / mM =Vs*[X]s / Vm*[X]m = Kx*Vs / Vm = CS /CM Vs is the volume of the stationary phase within the column and Vm is the volume of the mobile phase.

k= t'R / tM = (tRtM)tM
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Gaussian profile

All molecules of a given component do not move at an identical velocity. The centre of the elution band represents the average retention time of the analyte. Some molecules travel slower, some faster. This variation in velocity is a result of the number of sorption and desorption of the analytes and is what gives the detected signal its Gaussian profile.
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Theoretical plates

The number of theoretical plates (N) is a quantitative measure of the efficiency of a column and can be obtained directly from the chromatogram:
N = 16(tr/tw)

The theoretical plate model assumes the column to be made of a series of plates. The distribution of the analyte between the mobile and stationary phase occurs at each plate. Therefore, the higher the number of plates, the better the separation since more sorptiondesorption cycles occur.
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Column efficiency
Column efficiency can be expressed as Height Equivalent to a Theoretical Plate value (HETP or Hvalue): H = L/N This equation has the units of length since L corresponds to the column length. Thus, a column with a low H value is preferred to a high value.

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Separation / selectivity factor

The selectivity or separation factor (S) refer to a system's ability to distinguish between two components It is governed by the distribution of the analyte between the mobile and stationary phases. A and B correspond to two different analytes.

S = (trB-t0)/(trA-t0) = kB / kA

As S decreases towards a value of 1, the separation becomes more difficult. The only way to modify this parameter is to change one or both phases.
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Resolution

The resolution (R) is the degree of separation between two components. This kind of separation is essential in preparative chromatography where pure compounds are required.
R = 2 (trB - trA) / (tWA + tWB ) R = 1/4 N (S- 1/S) (k'/1 + k')

This expression shows that resolution is a function of three factors which can be adjusted independently: the column efficiency (N), the selectivity (S) and the capacity (k').
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Names

Symbol experimental quantity

of

Determined from

Migration species

time,

unretained

tM

Chromatogram

Retention time, species A and B Adjusted retention time for A Peak widths for A and B

(tR)A, (tR)B (t'R)A tWA, tWB.

Chromatogram (t'R)A = (tR)A - tM Chromatogram

Length of column packing Volumetric flow rate

Linear flow velocity Stationary-phase volume Concentration of analyte in

L F
u Vs CM, CS

Direct measurement Direct measurement

F and column dimensions Packing preparation data Analysis and preparation data

mobile ans stationary phase

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Necessary Formula

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Math Problem
Substances A and B have retention times of 16.40 and 17.63 min. respectively, on a 30.0-cm column. An unretained species passes through the column in 1.30 min. The peak widths (at base) for A and Bare 1.11 and 1.21 min. respectively. Calculate

a. b. c. d. e. f.

the column resolution. the average number of plates in the column, the plate height, the length of column required to achieve a resolution of 1.5. the time required to elute substance B on the column that gives an R, value of 1.5, And the plate height required for a resolution of 1.5 on the original 30-cm column and in the original time.
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Column resolution:

Here, trB = 17.63 , trA = 16.40, and

R = 2 (trB - trA) / (tWA + tWB )

tWA = 1.11, tWB = 1.21

then, R = 2 ( 17.63-16.40) (1.11+1.21) = 1.06

the average number of plates in the column : N = 16(tr/tw)2

NA = 16 ( 16.40/1.11)2 = 3493 NB = 16 ( 17.63/1.21)2 = 3397,

Naver = (NA+NB)/2 = 3445

C the plate height : H = L/N Here L = 30.0, then H = 30.0 / 3445 = 8.7 x 10-3 D the length of column required to achieve a resolution of 1.5. k and S do not change greatly with increasing N and L. Then, R1/R2 = N1/N2 or 1.06 /1.50 = 3445 / N2 or N2 = 6.9 x 103 and L = N2.H = 6.9 x 103 x 8.7 x 10-3 = 60 E the time required to elute tr1/tr2 = (R1/R2)2 then 17.63/tr2 = (1.06/1.50)2 Or tr2 = 36 min. Tr2/tr2 = (R1/R2)2 H1/H2 then H2= (R1/R2)2 H1 = 8.7 x 10-3 * (1.06/1.50)2
= 4.3

substance B on the column that gives an R, value of 1.5 f the plate height required for a resolution of 1.5

x 10-3
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Physical Forces and Interactions

Ionic Interactions

Ions of the same charge are repulsed whereas ions of opposite charge are attracted. These forces are long-range and relatively strong. Ionic interactions can also take place between ions.

Hydrogen Bonding
bond can be formed between molecules containing a hydrogen atom bonded to an electronegative atom like oxygen or nitrogen. Some can receive and donate a hydrogen atom. e,g. alcohols, amines and water. Hydrogen bonds are relatively strong forces.

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Van der Waals Forces: three types

Dipolar forces can be compared to magnetic forces. The dipole-induced dipole phenomenon can be compared to a magnet attracting nonmagnetic iron
is dependant on the polarisability of a molecule e.g. larger molecules generally have greater polarisability.

induced dipole-induced dipole, consist of weak forces that exist even in mono-atomic gases that are symmetrical and non-polar. Charge Transfer two molecules or ions combine by transferring an electron from one to the other. This process occurs mostly in gas chromatography
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Modes of Separation

Adsorption chromatography.
A solid stationary phase and a liquid or gaseous mobile phase are used. Solute is adsorbed on the surface of the solid particles. The more strongly a solute is adsorbed, the slower it travels through the column.

Partition chromatography.
A liquid stationary phase is bonded to a solid surface , silica (SiO2) in gas chromatography. Solute equilibrates between the stationary liquid and the mobile phase.

Ion-exchange chromatography
Anions such as SO3 or cations such as N(CH3) 3 are covalently attached to the stationary solid phase, usually a resin. Solute ions of the opposite charge are attracted to the stationary phase. The mobile phase is a liquid.

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Size exclusion chromatography.

this technique separates molecules by size, with the larger solutes passing through most quickly. the liquid or gaseous mobile phase passes through a porous gel. Large molecules stream past without entering the pores. Small molecules take longer to pass through the column because they enter the gel.

Affinity chromatography.
It employs specific interactions between one kind of solute molecule and a second molecule that is covalently attached to the stationary phase. The immobilized molecule might be an antibody to a particular protein. When a mixture containing a thousand proteins is passed through the column, only the one protein that reacts with the antibody binds to the column.
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Stationary Phases versus Mobile Phases

The mobile phase can be

a liquid (as in LC) or a gas (as in GC).

the stationary phase can be a liquid or a solid.

In effect, there are three stationary phase configurations:
a solid packed into a column (as in SEC), a solid coating the surface of a flat, plane material (as in thin layer chromatography - TLC) and a liquid coated on the inside wall of an open tube (LLC).
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Application : chromatography

It can be employed for qualitative identification and quantitative determination of separated species.
Qualitative Analysis Quantitative Analysis

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Qualitative Analysis
A chromatogram provides only a single piece of qualitative information about each species. Chromatograms can be used to derive additional data. Spectral wavelength can not be revealed by chromatography, unlike by a single IR, NM resonance, or mass spectrum.

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Quantitative Analysis

a comparison of either the height or the area of the analyte peak with that of one or more standards.
Analyses Based on Peak Height Analyses Based on Peak Areas Calibration and Standards The Internal-Standard Method The Area-Normalization Method

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