Theoretical Plate

A theoretical plate is a hypothetical zone found in a separation process in which two

phases, such as liquid and vapor, have established a state of equilibrium with one
another.

From: Methods in Enzymology, 2013

Related terms:

Facilitated Diffusion, Mobile Phase Composition, pH, Distillation, Solute

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Learn more about Theoretical Plate

Unit Operations
Pauline M. Doran, in Bioprocess Engineering Principles, 1995

10.7.3 Theoretical Plates in Chromatography

The concept of theoretical plates is often used to analyse zone broadening in
chromatography. The idea is essentially the same as that described in Section 10.4 for
an ideal equilibrium stage. The chromatography column is considered to be made
up of a number of segments or plates of height H; the magnitude of His of the
same order as the diameter of the resin particles. Within each segment equilibrium
is supposed to exist.

As in adsorption operations, equilibrium is not often achieved in chromatography

so that the theoretical-plate concept does not accurately reflect conditions in the
column. Nevertheless the idea of theoretical plates is applied extensively, mainly
because it provides a parameter, the plate height H, which can be used to characterise
zone spreading. Use of the plate height, which is also known as the height equivalent
to a theoretical plate (HETP), is acceptable practice in chromatography design even
though it is based on a poor model of column operation. HETP is a measure of zone
broadening; in general, the lower the HETP value the narrower is the solute peak.
HETP depends on various processes which occur during elution of a chromatogra-
phy sample. A popular and simple expression for HETP takes the form:

(10.44)

where H is plate height, u is linear liquid velocity, and A, B and C are experimental-
ly-determined kinetic constants. A, B and C include the effects of liquid–solid mass
transfer, forward and backward axial dispersion, and non-ideal distribution of liquid
around the packing. As outlined in Section 10.6.4, overall rates of solute adsorption
and desorption in chromatography depend mainly on mass-transfer steps. Values of
A, B and C are reduced by improving mass transfer between liquid and solid phases,
resulting in a decrease in HETP and better column performance. Eq. (10.44) and
other HETP models are discussed further in other references [16, 17].

HETP for a particular component is related to the elution volume and width of the
solute peak as it appears on the chromatogram. If, as shown in Figure 10.18(a), the
pulse has the standard symmetrical form of a normal distribution around a mean
value, the number of theoretical plates can be calculated as follows:

Figure 10.18. Parameters for calculation of: (a) number of theoretical plates, and (b)
resolution.
(10.45)

where N is number of theoretical plates, Ve is the distance on the chromatogram

corresponding to the elution volume of the solute, and w is the base line width of the
peak between lines drawn tangent to the inflection points of the curve. Eq. (10.45)
applies if the sample is introduced into the column as a narrow pulse. Number of
theoretical plates is related to HETP as follows:

(10.46)

where L is the length of the column. For a given column, the greater the number of
theoretical plates the greater is the number of ideal equilibrium stages in the system
and the more efficient is the separation. Values of H and N vary for a particular
column depending on the component being separated.

> Read full chapter

Capillary Zone Electrophoresis

Robert Weinberger, in Practical Capillary Electrophoresis (Second Edition), 2000

2.9 OPTIMIZING THE CAPILLARY LENGTH

The efficiency of the separation (theoretical plates) is directly proportional to the
capillary length (38), provided the field strength is kept constant. The limitation
here is available voltage. Most instruments produce a maximum of 30 kV. Once the
capillary length reaches a certain point, the field strength must be reduced and no
further gains in efficiency are realized (6). Based on Eq. (2.16), the electrophoretic
resolution depends on the square root of the number of theoretical plates and,
thus, on the square root of the capillary length (38). Increasing the capillary length
beyond the limits imposed by the voltage maximum lengthens the separation time
without any substantial benefits. These effects are illustrated in Figure 2.22, where
the number of theoretical plates is linear with the capillary length until 30 kV is
reached. Note that the resolution increase is proportional to the square root of the
number of theoretical plates.
FIGURE 2.22. Impact of capillary length on the number of theoretical plates and
resolution.

Most chemists overly rely on the length of the capillary to perform their separations.
This results in lengthy separations. Since diffusion is time related, the sensitivity
of the method declines as well. If more time is spent optimizing the separation
chemistry, shorter capillaries can be employed, with obvious benefits.

> Read full chapter

Bioseparation Engineering
E.K. Lee, S.J. Ahn, in Progress in Biotechnology, 2000

5.1 Column Packing

For ion exchange column, HETP (height equivalent to a theoretical plate) is mea-
sured usually by conductivity spiking method and its value is compared against the
mean bead diameter [5]. If it is lower than approximately twice the mean diameter, a
column is considered well packed. HETP needs to be measured after each packing.
It is essential to use the same HETP measurement method each time and to trace
its value after each cycle. It should be noted that HETP can only indicate how well
packed the bed is and not the condition of the resin [7].

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Gas Chromatography and Gas Chro-
matography—Mass Spectrometry
Eric Stauffer, ... Reta Newman, in Fire Debris Analysis, 2008

8.2.4 Efficiency
The efficiency of a column is measured by the number of theoretical plates (N), or
more often, the height equivalent to a theoretical plate (HETP or H). The theoretical
plate number (N) usually is expressed by one of two equations [9]:

(Equation 8-2)

or

(Equation 8-3)

where tR refers to the retention time of the peak and Wb refers to the peak width
at baseline in Equation 8-2 and Wh its width at half-height in Equation 8-3. Figure
8-3 illustrates the values used with these equations. The higher the plate number
N, the greater the efficiency of the column. One can understand quickly that the
narrower the peak (low W), the higher N, and thus the efficiency. Efficiency is
thus a measure of peak broadening; an efficient system will result in narrow peaks,
whereas a less efficient column will contribute to band broadening.

FIGURE 8-3. The different values used to calculate the plate number (N).

The height equivalent to a theoretical plate (HETP) can be calculated when both N
and the column length (L) are known [10, 11]:

(Equation 8-4)

As a result, the lower the HETP, the better the resolution and the more efficient the
separation. Efficiency is optimized when N is maximized and HETP is minimized.
The van Deemter equation is another way of expressing efficiency; it takes into
account three factors that, along with the mobile phase linear velocity, will affect
efficiency and, therefore, the HETP value [13]. These factors are represented by the
variables A, B, and C in the van Deemter equation [10]:

(Equation 8-5)

Theoretical Plate Concept

Historically, the concept of the number of plates as a measure of efficiency is based

upon separation by distillation. The ability to separate by distillation was reflected in
the number of plates, within each of which distinct equilibria occurred. The greater
the number of plates, the better the potential for separation. Imagine a distillation
column with two plates, then one with 100 plates (refer to Figure 7-4, which shows a
simplified schematic of a distillation tower with only five plates). Clearly, more plates
allow for improved separation as a more refined gradient of temperatures can be
used. This concept was adapted to chromatography by Martin and Synge in 1941
[12]:

The behaviour of a column consisting of a number of “theoretical plates”, within

each of which perfect equilibrium between the two phases occurs, can be described
with great simplicity. Peters [1922] showed that the continuous or packed type of
distillation column (in which equilibrium is not established at any point) could be
divided up into a number of layers each of which was equivalent to one theoretical
plate, and the height of such a layer was called the H.E.T.P. or “height equivalent to
one theoretical plate”. For the present purpose the H.E.T.P. is defined as the thickness
of the layer such that the solution issuing from it is in equilibrium with the mean
concentration of solute in the non-mobile phase throughout the layer. It can be
shown from diffusion arguments that the H.E.T.P. is a constant through a given
column except when the ratio of the concentrations of the solution entering and
leaving the plate differs greatly from unity [cf. Sherwood, 1937]. It may be taken as
constant for the chromatogram without serious error.

The theoretical plate concept is a good way to express relative efficiency, although
it is important to understand that this model has limitations. When using the term
“theoretical plates” one must remember that there are not actual distinct equilibria
being achieved. Another concern with the theoretical plate model is that it does
not adequately account for band broadening. However, the number of theoretical
plates or the HETP is still used today as a means for expressing the efficiency of a
chromatographic system.

in which A represents the Eddy diffusion phenomenon2 in the column, B represents

molecular (or axial) diffusion, C relates to the resistance to mass transfer, and μ is
the linear velocity of the mobile phase through the chromatographic system [10].
The linear velocity is calculated based on the following equation [15]:
(Equation 8-6)

where L is the length of the column [cm] and tM is the retention time[s] of an
unretained analyte (dead time). The mobile phase linear velocity, μ, therefore is
expressed in cm/s.

Using the van Deemter equation, one can plot the efficiency of a carrier gas in
terms of HETP as the dependent variable (y-axis) against the linear velocity as the
independent variable (x-axis). This plot is referred to as a van Deemter plot, and can
be used to compare the efficiency of one carrier gas to another (see Figure 8-4).

FIGURE 8-4. A van Deemter plot showing the effect of each variable from the
corresponding equation. Note that the Eddy diffusion is independent of the linear
velocity, thus it is constant. Because the resistance to mass transfer (C) is multiplied
by the linear velocity, it increases with μ. The invert occurs with the molecular
diffusion (B).

All three parameters (A, B, and C) lead to band broadening: the spreading apart of a
group of identical analytes throughout the system. By minimizing band broadening,
identical analytes stick together and a better separation is obtained because peaks are
narrow rather than broad. With Figure 8-4, one can quickly appreciate the influence
of the mobile phase flow rate on each parameter. The A term in the van Deemter
equation is relevant only to packed column systems and thus is constant for a given
column. Nevertheless, it is independent of the linear velocity μ as indicated by
Equation 8-5 and Figure 8-4. The B term represents the natural tendency of the
analytes to redistribute themselves from a region of high concentration to a region
of lower concentration in the mobile phase [2]. Finally, the C term, resistance to
mass transfer, represents the fact that the transfer of an analyte from the mobile to
the stationary phase and vice versa is not instantaneous and has a certain inertia. As
a result, the analyte concentration profile of the stationary phase is slightly behind
the equilibrium position and the analyte concentration profile of the mobile phase
is slightly ahead of the equilibrium position [2]. As expected, the C term increases
with the flow rate as shown in Figure 8-4.
Since the Eddy diffusion does not apply to capillary columns, it may be useful to
become familiar with the Golay equation, in which the A term has been removed
and the C term has been expended [8]:

(Equation 8-7)

where Cs is the mass transfer from the stationary phase to the mobile one and Cm
is the mass transfer from the mobile phase to the stationary one.

Another important chromatographic concept is that of resolution. The resolution, R,

is a measure of the true separation of two consecutive peaks [10]. One can calculate
the resolution of a chromatographic system from two consecutive peaks using the
following equation:

(Equation 8-8)

where d is the separation between two peaks (measured from top to top) and W1 and
W2 are the widths at baseline of the two peaks. This is illustrated in Figure 8-5.

FIGURE 8-5. The measures used to calculate the resolution (R) of a chromatographic
system.

The goal in chromatography is to avoid coelution. This is an issue because two

coeluting peaks will mix together in the detector. For example, each compound's
contribution to the overall structural information provided by the mass spectrometer
will not be distinguishable, thus their identification will be hampered. This is the
reason why it is important to achieve baseline resolution (R = 1.5) as the ultimate
goal for all peaks. In fire debris analysis, this goal can never be achieved in practice,
however it is important to adjust the chromatographic parameters to approximate
it as closely as possible. A resolution below 1.5 will not provide complete separation
and a resolution above 1.5 does not offer any extra advantages to the separation.
Figure 8-6 shows the peak configurations for three different values of R.
FIGURE 8-6. The effect of resolution (R) on the separation of two consecutive peaks.

> Read full chapter

Capillary Zone Electrophoresis

Robert Weinberger, in Practical Capillary Electrophoresis (Second Edition), 2000

C. A BASIS FOR METHODS DEVELOPMENT AND OPTIMIZA-

TION
The remarkable ability of electrokinetic chromatography to provide a few hundred
thousand theoretical plates permits complex separations to be performed, frequently
without the need to employ chemometrics-based optimization schemes. Following
a scheme such as that illustrated in Figure 4.28 will usually lead to an adequate
separation, often during the course of less than a day of experimentation. More
often than not, the 100 mM SDS, 20 mM borate buffer will yield a good starting
point.
FIGURE 4.28. An empirical scheme for methods development using MECC.

Switching to surfactants other than SDS is normally beneficial for the separation
of nonpolar substances or when SDS alone gives too much or too little retention.
Even in the former case, the use of cyclodextrins permits the separation of nonpolar
species such as aromatic hydrocarbons. Usually, alternative surfactants should be
considered only after other experiments covering pH, modifiers, and so forth have
been performed using SDS—unless, of course, a suitable reference has been located.
Most problems will be solvable using SDS or SDS with various additives. Even if
a publication reported on an alternative surfactant system, the separation may be
possible with SDS. Often, several different surfactant systems are suitable for a given
problem.

On the other hand, bile salts are useful for separating rigid planar molecules such
as steroids (131). The sterol architecture resembles the steroid structure; thus, the
old adage from freshman chemistry, “like dissolves like,” applies here. In a similar
fashion, the planar macrolide antibiotics are well separated using bile salt surfactants
(105). In any event, a few scouting runs should point you in the correct direction.

Once the appropriate system has been identified through scouting runs, opti-
mization is usually straightforward. Adjustments in pH and additive concentrations
should be carefully studied. It is best not to rely on a long capillary to perform
the separation, although this may become necessary when many components are
being resolved. In complex samples, solving the separation of a pair of overlapping
components often causes coelution of other solutes.
For these situations, statistical tools can be a valuable tool for speeding meth-
ods development. This can be particularly true when ternary blends of solvents
or cyclodextins are needed to optimize the separation. For example, overlapping
resolution maps (ORM) have been used for years to optimize HPLC separations. In
1991, this technique was used to optimize the separation of plant growth regulators
using mixtures of -, -, and -CD (132), and in 1997, it was applied to optimize the
separation of quinolone antibacterials using cholate and heptane sulfonate (133). A
pure chemometric approach using Plackett-Burman statistics has also been shown
useful in the separation of testosterone esters (134). While these tools are not often
used, they should be considered when trial and error proves frustrating.

> Read full chapter

Appendix 12a. Nomenclature | Chro-

matography (IUPAC Recommenda-
tions 1993)
L.S. Ettre, in Encyclopedia of Separation Science, 1993

3.10.06 Effective plate height (Heff)

The column length divided by the effective plate number:

It is also called the Height Equivalent to One Effective Plate.

Notes: In the former literature the expression ‘height equivalent to one effective
theoretical plate’ had been used to express this term. This is incorrect, since the plate
height is either theoretical or effective (see 3.10.04), but cannot be both.

In former nomenclatures the respective symbols h and H have been used for the
plate height and the effective plate height, respectively. However, there was often a
confusion in the proper selection of lower case and capital letters and also due to
the fact that h (lower case letter) is also used to express the reduced plate height (see
3.10.07). The present usage is suggested in order to avoid any confusion.

> Read full chapter

VARIABLES IN THE GAS CHRO-

MATOGRAPHIC PROCESS
WALTER JENNINGS, ... PHILIP STREMPLE, in Analytical Gas Chromatography (Sec-
ond Edition), 1997

5.22 Chromatographic Efficiency under Temperature-Pro-

grammed Conditions
Despite the temperature dependence of various factors affecting the height equiv-
alent to a single theoretical plate in both the Golay and van Deemter equations,
the width of any particular chromatographic peak in the absence of significant
“extracolumn effects” will ultimately depend on how fast the solute corresponding to
that peak exits the column and enters the detector. Consequently, accurate estimates
of chromatographic efficiency for a particular solute can typically be obtained
from Golay or van Deemter equations in which various temperature-dependent
parameters assume values suggested by the temperature at which the same solute
elutes under temperature programmed conditions (i.e., TR). This is particularly true
whenever the pressure drop across the column is small.

Although perhaps not intuitively obvious, this can be best understood by recalling
the temperature dependence of k [Eq. (5.49)] and the nature of Kc as an equilibrium
distribution constant [Eq. (5.44)]. As the temperature increases under tempera-
ture-programmed conditions, solute retention factors decrease and corresponding
solute equilibrium distribution constants favor higher solute concentrations in the
mobile phase. As a result, the rate at which solutes elute off the stationary phase and
are swept out of the column is enhanced and the chromatographic efficiency of
the column appears to improve.

Conversely, if the temperature is caused to decrease as a function of time during a

temperature-programmed run, solute retention factors become larger and equilib-
rium distribution constants favor lower solute concentrations in the mobile phase.
This impedes the rate at which solutes can be swept out of the column and so the
chromatographic efficiency of the column appears to degrade.

When the pressure drop across the column is large, variation in the linear velocity of
carrier gas as a function of distance from the column inlet [Eq. (5.12)] can become
extreme. This complicates matters somewhat by requiring the need to consider the
degree to the height equivalent to a single theoretical plate at Tr may gradually
become a function of the linear velocity of carrier gas near the outlet rather than
the average linear velocity of carrier gas through the column as a whole.

> Read full chapter

Polymer Characterization
John V. Dawkins, in Comprehensive Polymer Science and Supplements, 1989

12.3.1 Plate Height and Plate Number

A measure of the efficiency of a chromatography column is the height equivalent
to a theoretical plate or plate height H.70 The plate height for an experimental
chromatogram is calculated from the expression

(14)

where L is the column length and N is the plate number. If the chromatograms
such as the peaks in Figure 1 are symmetrical, corresponding to a normal error (or
Guassian) function, then N may be determined from

(15)

where w0.5 is the width of the chromatogram at half its height. A typical micropartic-
ulate packing with particle diameter  10 μm will generate a HPSEC column having
N > 20 000 plates m−1 for a solute eluting at VR = Vo + Vi.

> Read full chapter

Bioseparation Engineering
Oliver Kaltenbrunner, ... Shuichi Yamamoto, in Progress in Biotechnology, 2000

2 THEORY
The term used to measure dispersion in a chromatographic column is the height
equivalent of a theoretical plate (HETP) and can be defined as.

Equation 1

The most common approximation assumes a normal distribution used is based on

the retention time of the peak maximum (tc) and the peak width at half height (wh)

Equation 2

Band broadening as a function of flow velocity (u) can be described by the Knox
equation(11) as,

Equation 3
where the reduced plate height is the plate height relative to the particle diameter
(dp)

Equation 7

and the group ReSc is the dimensionless velocity The terms a, b, and c of Equa-
tion 3 represent the band broadening contributions from axial dispersion, diffusion,
and mass transfer limitations, respectively. The description of the deviation of the
response profile from its ideal symmetrical shape can be simply described by the
asymmetry factor (Af) which is the ratio of half widths of the peak at 10 % peak height.
A symmetrical peak gives Af = 1. Af < 1 indicates fronting peaks and Af > 1 indicates
tailing peaks

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PERFUSION CHROMATOGRAPHY: A
NOVEL TOOL FOR PROTEIN PURIFI-
CATION AND ANALYSIS
Noubar B. Afeyan, ... Fred E. Regnier, in Techniques in Protein Chemistry III, 1992

V IMPLICATIONS OF FLOW-THROUGH PARTICLES ON

CHROMATOGRAPHIC PERFORMANCE
In standard diffusion-based media, as flow rates increase, the bandspreading (or the-
oretical plate height) increases linearly (i.e. chromatographic efficiency decreases
linearly). This classical behavior is due to the so-called “C-term” in the VanDeemter
equation, which accounts for mass transport into and out of the particle. With
Perfusion Chromatography media, this relationship between flow rate and band-
spreading no longer applies. Once the rate of perfusive transport exceeds that of
diffusion (typically below 300 cm/hr linear velocity), bandspreading becomes virtually
independent of flow rate, up to at least 7000 cm/hr linear velocity4–8 (figure 2). This
contrasts sharply with the significant increase in bandspreading that occurs with
conventional media. A comparison between a conventional 300Å Reversed-Phase
separation of proteins with POROS perfusable supports is shown in figure 3. Clearly,
the high speeds associated with the separation are not compatible with conventional
supports. An additional effect of convective Flow through the particle is that the plate
height or bandspreading becomes dependent only upon the particle diameter, and
not the particle diameter squared, as with conventional diffusive supports 5. This is
shown in Figure 2, where 10 and 20 µm perfusive supports show the same reduced
plate height (plate height divided by particle diameter), which remains constant with
flow rate. This is especially important for large scale preparative applications, since
it allows very high resolution to be achieved with relatively large particles that give
low pressure drop at high flow rate.

Figure 2. Reduced plate height vs. linear velocity for conventional (10 μm, 300 Å) and
Perfusion Chromatography media of various particle sizes. All columns are polymer-
ic reversed-phase and were tested with insulin under non-retained conditions.

Figure 3. Comparative analytical separations with conventional perfusable supports.

3A shows the separation of a protein mixture on a 300Å polymeric reversed phase
column at 1800 cm/hr. Figure 3B shows the same separation on a POROS column at
the same speed while 3C shows the POROS run at 360 cm/hr (typical of conventional
HPLC).

A:
Column: PLRP-S 300Å 4.6mmD/50mmL
B,C:
Column: POROS R 4.6mmD/100mmL
Sample: 1 Ribonuclease
 2 Lysozyme
3 ß-Lactoglobulin A,B
4 Ovalbumin
Eluent: 0.1% TFA/water
0.1% TFA/acetonitrile
Gradient: 0-70% in 3 column volumes
Flowrate: A − 5ml/min
B − 5ml/min
C − 1ml/min

In preparative chromatography, throughput (amount of product produced per unit

time) is as important as resolution. One important variable affecting throughput
is the dynamic loading capacity, normally measured by applying a sample feed
continuously to the column at a given flow rate until a significant concentration
is detected in the eluent (“breakthrough”). The amount of feed bound divided by
the column volume gives the unit dynamic loading capacity. This technique is called
frontal chromatography.

Dynamic capacity, as measured by frontal chromatography, is expected to have a

dependence on flow rate. It has been shown that with “diffusion-limited” media,
increasing the loading flow rate causes substantial loss in dynamic capacity as feed
molecules begin emerging prematurely from the column due to slow mass trans-
port.4,11 Perfusion media have virtually the same dynamic capacity as conventional
supports at very low flow rates but show extremely sharp breakthrough curves with
no change in dynamic capacity over a very broad flow rate range1–3,3, 5 (Figure 4).

Figure 4. Dynamic binding capacity vs. linear velocity for conventional and Perfusio
nChromatography media. The conventional support (fast-flow agarose, 50-100 μm)
and perfusive support (20 μm) were both strong anion exchangers. Capacity was
measured by frontal analysis using BSA as the test solute.
 Table 1 summarizes the relative merits of each of the available approaches to
chromatographic mass transport.

Table 1.

Speed Capacity Resolution Scalability Pressure

Low Pressure − + − + Low
LC
Conventional +/− + + +/− High
HPLC
Non-Porous + − + − Very High
HPLC
Membranes + − − + Low
Perfusion + + + + Low-High

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