lecture 21 intro to chrom
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8/8/2019 Lecture 21 Intro to Chrom
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Lecture #21 Introduction to Chromatographic Separations
Reading: Chapter 26, page 762 – 787; Problems: 26-1,2,5,6,7,10.
• overview and general description of chromatographic methods
• migration rates of solutes in column;
• column efficiency;• optimization of column performance;
• quantitative application of chromatographic methods.
8/8/2019 Lecture 21 Intro to Chrom
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Overview of chromatography
Before the middle of 20th century,Analytical separations were carried out by classical methods, such as
precipitations, distillation, extraction, which are of course still useful for today’s
lab work.
• Separation is a key aspect of every analytical methodology!• A real world sample contains many analytes that are responsive to the particular
perturbation.• Most analytical methods do not offer sufficient selectivity to be able to
differentiate all the analytes that might be present.• Most methods involve separation of the analytes first, followed by detection.
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• Mobile phase --- in which the solutes are transported. It can be gas, liquid,
or supercritical fluid.
• Stationary phase --- through which the mobile phase is forced to flow. It
should be immiscible with the mobile phase.• Different species have different distribution between the mobile and
stationary phases, and thus can be separated when being transported along
the mobile phase.
• Stationary phase can be supported on flat plate (planar chromatography),or held in a narrow tube (column chromatography).
• We focus on column chromatography, which is more popularly used.
Depending on the mobile phase, three kinds of column chromatography
methods are available:
Gas chromatography, liquid chromatography, supercritical-fluid
chromatography.
General description of chromatography
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Elution chromatography on columns
Figure 26-1.
diagram showing the separation of
a mixture of components A and B
by column elution. Bottom: the
output of the detector signal at the
various stages of elution.
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Chromatogram: effects on separation efficiency
The longer the analytes (solutes) stay with the stationary phase, the better the
separation efficiency is, but the broader (in time) the peaks get .
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Basics of chromatography
• Equilibrium between mobile and stationary phases --- K = CS /CM
K is the distribution constant (partition coefficient), CS is the concentration of solute
in mobile phase; CM is the concentration in stationary phase.
• Efficiency of separation --- N = L/H = (4tR /W)2
N is the number of plates; N is the length (in cm) of the column; H is the plate height.
A pseudo-equilibrium is established onthe column as the analyte develops a
distribution within the mobile phase andthe stationary phase.
2 2
2
16
4
R
R
LW H
Lt
LW
t
σ
σ
= =
= See next slide for W,the peak bottom width.
6
tR
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Factors affecting peak width (plate height)
• The narrower the peak, the smaller the plate height (the better the separationefficiency).
• Van Deemter Equation: Plate height, H = A + B/u + Cu
u is the linear mobile-phase velocity; u = L/tM
A, B, C are the coefficients related to the phenomena of multiple flow paths,longitudinal diffusion, and mass transfer between mobile and stationary phases,
respectively.
9
B/u
A process driven by
concentration difference
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Factors affecting peak width
(insight of van Deemter equation)
10
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Factors affecting peak width
(effect of column particle size)
11
The smaller the particle size, the more uniform the column packing, then themore tolerant to the change in mobile-phase velocity.
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Factors affecting peak width• An ideal chromatography should have the shortest retention time and have
adequate resolution to separate all closely eluting components.
• The smaller plate height, the better the resolution (Rs) is. The optimized value ofplate height can be obtained experimentally following Van Deemter equation.
Rs=2(trB-trA)/(WA+WB)
8
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General Elution Problem
• Quite often, a constant elution condition does not result in a satisfied separationas shown in the Figure.
• To solve this problem, the separation conditions are often not kept constantduring the entire run, but rather are altered at some point during the run.
• Typical separations often begin with one set of conditions and end with a different
set.• Variation in mobile phase composition is a widely used strategy to optimize the
separation --- gradient elution . To be discussed in HPLC.
5
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Quantitative application of chromatography
• Not enough for qualitative analysis, since the only experimentally determined
parameter is the retention time, which can only tell the presence of multiplespecies, but cannot confirm which is which.
• Chromatography is often combined with MS for qualitative analysis.
• Very good for quantitative analysis --- based on peak height or peak area. Peak
area measurement often gives more reliable result, since the peak height isdependent on the broadening effects as mentioned above.
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