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Chromatography: Fundamentals and Applications

Chromatography: Fundamentals and Applications

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PRINCIPLES AND APPLICATIONS Ricón Ullate Rafael Alonso Gómez-Serrano Alvarez
1. Chromatography Basics

Before defining the basic concepts to understand the different types of chromato graphic separations, make a brief introduction and an overview on chromatography . The concepts are explained below refer to the column chromatography, however, it is important to note that as equilibria underlying the two types of chromatog raphy are identical,€the theory to the column chromatography is also adapted eas ily to flat-bed chromatography.

1.1. Brief introduction to chromatography
Chromatography is a widely used in all branches of science and allows the separa
tion, identification and determination of chemical components in complex mixture
s. No other method of separation is so strong and so general application such as

1.2. Overview chromatography
It is difficult to rigorously define the term Chromatography€because that name h
as been applied to various systems and techniques. However, all these methods ha
ve in common the use of a stationary phase and a mobile phase. In all chromatogr
aphic separations, the sample is moving with a mobile phase, which can be a gas,
liquid or supercritical fluid. This mobile phase is passed through a stationary
phase which is immiscible with, and attached to a column or a solid surface. Th
e two phases are chosen in such a way€that the components of the sample are dist
ributed differently between the mobile phase and stationary phase. Those compone
nts are strongly retained by the stationary phase move slowly with the flow of t
he mobile phase, on the contrary, the components that bind weakly to the station
ary phase, they move quickly. Due to the different mobility of the sample compon
ents separate into discrete bands or zones that can be analyzed qualitatively an
d / or quantitatively.

1.3. Basics
Chromatogram: If we place a detector at the end of the column that responds to t
he concentration of solute and its signal is recorded versus time (or volume of
mobile phase added) gives a series of peaks representing a graph called the chro
matogram. This graph is useful for both qualitative and quantitative analysis. T
he position of the peaks in the time axis may serve to identify the components o
f the sample, the areas under the peaks provide a quantitative measure of the am
ount of each component (see Figure 4-1).€TR Retention time: The time that elapse
s after the sample injection until the peak concentration of the analyte reaches
the detector is the time it takes for a compound to emerge from the column is t
he time it takes to receive the maximum peak (Figure 1-1).

1It represents the symbol tR. The average linear velocity v is the solute migrati

L tR
where L is the length of the column is filled.
Figure 1-1. Characteristic chromatogram of a mixture of two components.€The smal

l peak on the left represents a species that is not retained in the column, and

thus reach the detector almost immediately after the start of elution. Therefore , its retention time tM is approximately equal to the time taken for a molecule of the mobile phase to pass through the column.

TM Downtime: The time necessary for the species reaching the detector is not ret
ained, is the migration time of the species not retained, it is the time require
d for, on average, one molecule of the mobile phase passes through column,€(Figu
re 1-1). Similarly the average linear velocity or the movement of molecules in t
he mobile phase is
u = L tM

Constant pattern: In general, the distribution equilibria involved in chromatogr
aphy are described by simple equations involving the transfer of an analyte betw

een the stationary and mobile phases. Thus, for the solute A, we can write
⇔ removability Aestacionaria
The equilibrium constant K for this process is called distribution constant, the

distribution ratio or partition coefficient,€and measures the distribution of a
nalyte between the stationary phase and mobile phase. It is defined as:
Mwhere CS is the molar concentration of solute in the stationary phase and CM is

the molar concentration in the mobile phase.
CS = moles / liter of analyte in the stationary phase. CM = moles / liter of ana
lyte in the mobile phase. Retention factor or capacity factor:

2This parameter describes the rate of migration of the solutes (analytes) in the
column. For a species A, the capacity factor is defined as k'a
'AVS kA = K t-t', kA = R M tM VM

where KA is the distribution coefficient of species A, VS is the volume of the s tationary phase and VS is the volume of mobile phase. When the capacity factor f or a species is much smaller than unity, the elution takes place so rapidly that it is difficult to accurately determine the retention times, these are too shor t. However when the retention factor is about 20 or 30 or maybe more, the retent ion times are too long (Figure 1-1).€Sélectivité Factor: The selectivity factor

α in column for two species A nd B is defined s:

(T) - KB t k 'α = B α = RBM' kA KA (tR) A - tM
where KB is the distribution coefficient for the more strongly ret ined species

B, nd KA is the distribution coefficient for the le st ret ined, or more r pidl
y eluted, the species A. According to this definition α is lw ys gre ter th n u

nity, th t B is ret ined compound (of> tR). k'B yk'B where re the c p city f ct ors of B nd A, respectively.€Resolution Chrom togr phy: The chrom togr phic res olution of column RS is qu ntit tive me sure of its bility to sep r te two n lytes. The resolution of column is defined s

2 ⎡ (tR) - (tR) A ΔZ ⎤ ⎦ 2 ⎣ B RS = RS = WA WA WB WB
where WA nd WB re the widths of the pe ks A nd B.

From Figure 1-2 it follows th t if R> 1.5 the sep r tion of the components is co mplete, not if R <1.5. So for given st tion ry ph se, the resolution c n be im proved by lengthening the spine, incre sing the number of pl tes.€Although neg tive consequence of incre sing food is the incre se of the time required for se p r tion.

3Figure 1-2. Sep r tions for three different resolutions. Where Rs = 2ΔZ / (WA WB

Chrom togr phy Efficiency: Efficiency is defined by two terms, which re qu ntit tive me sures of the efficiency of column: 1 pl te height H nd 2nd pl te num ber N. The two re rel ted by the equ tion:

where L is the length (usu lly in centimeters) of the filling of 1st column. Fro
m Equ tion (1.8) th t the pl te height H is:

The chrom togr phic column efficiency incre ses with the number of dishes, nd t
he sm ller the pl te height. There re differences in the efficiencies due to di
fferences in the type of column nd / or the type of mobile nd st tion ry ph se
s. Efficiencies in terms of number of dishes r nging from hundreds to thous nds,
nd pl te heights of few tenths of thous ndth of centimeter or less. Ther

e is nother equ tion for c lcul ting the number of pl tes N:
⎛ ⎞ N t = 16 W ⎜ ⎟ ⎝ ⎠ R

In this c se N c n be c lcul ted from two me sures of time, tR, W (pe k width) t
o c lcul te H, h de lso know the length of the column p cking L. (Figure 1-3)
4Figure 1-3. Defining the number of pl tes N = 16 (tR / W) 2

It is gener lly ssumed th t the chrom togr phic b nds h ve G ussi n sh pe, so it is convenient to define the efficiency of column in terms of v ri nce per unit length of the column. Thus, the pl te height H is given by:


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