Polytechnic University of Bucharest

Faculty of Engineering in Foreign Languages

Chemical Engineering Department



Breakthrough Curves
Determination of Specific Parameters



student: Eugen S. Andreiadis
group: 1254E / 2004 - 2005
coordinator: as. ing. Maria Mihaly



2.

ntroduction


olid-phase extraction (SPE) is a simple, fast technique for the clean up and isolation of
analytes from complex matrices. It has many advantages over more traditional extraction
and clean-up procedures such as high recovery rates, the ability to extract multiple
components from one sample and, most importantly, the components of interest are isolated
in a highly pure, concentrated form.

In addition, the SPE method allows for the quick and simple analysis of the properties of
various solid phases, as regards the number of theoretical plates, the linear capacity, the
recovery factor or the type of adsorption, to name but a few. The present paper aims to
illustrate this method of analysis, by presenting the results obtained for the commonly used
C18-bonded silica phase, as well as the results obtained by testing new types of materials,
prepared using two different matrixes.



heoretical Aspects

2.1. The Breakthrough Curve

he breakthrough curve (BC, also known as frontal chromatogram) represents the
evolution of the solution concentration in function of adsorption parameters like contact
time between liquid and solid phase, solvent concentration and temperature (see Figure 1).
















Figure 1. A typical breakthrough
curve and the fundamental
chromatographic parameters

I
S
T
T
VB VR
VE V0
Volume of the effluent
C
o
n
c
e
n
t
r
a
t
i
o
n

o
f

t
h
e

a
n
a
l
y
t
e

c0
n
adsorbed
n
lost
1.
2.

3.
In the case shown in the figure, the total amount of the analyte originally present in the
sample is the product of the analyte concentration and sample volume, c0 V0. The amount
lost (nlost) due to breakthrough is graphically represented by the area under the frontal
chromatogram. Thus, the amount retained in the SPE column (nadsorbed) equals the difference
between the total amount and the amount lost, graphically corresponding to the area above
the frontal chromatogram.

The breakthrough curve can give precious information regarding the cartridge and the solid
phase inside, like the number of theoretical plates, the linear capacity or the recovery factor.

2.2. Methods for BC parameters evaluation

everal fundamental parameters characterize the curve: the breakthrough volume VB, the
equilibrium volume VE, the retention volume VR and the total volume V0.

The precise determination of these parameters is subject to debate. Following, we shall
present and compare two methods found in literature for the evaluation of the parameters
and an original method based on the extreme points of the third and first derivative curve.

Standard deviation method

Using this method the retention time of the analyte (VR) can be determined as the value
corresponding to half the initial concentration [1], as indicated in Figure 2.
VR = 0.5 c0 (1)











Figure 2. Determination of
the parameters using the
standard deviation method


The breakthrough volume (VB) is usually defined as:
VB = VR - 2 V (2)
where V is the standard deviation of the derivative curve which can be determined
graphically from the breakthrough curve as shown in Figure 2. V is in direct relation to the
S
VB VR
VE V0
Volume of the effluent
C
o
n
c
e
n
t
r
a
t
i
o
n

o
f

t
h
e

a
n
a
l
y
t
e

c0
0.5 c0
0.841 c0
0.159 c0
V V*

4.
efficiency of the SPE column, that is, the number of theoretical plates (N) which can be
calculated from the breakthrough curve using the equation:

( )

=
R R V
2
V
V V
N (3)
Analogously, the equilibrium volume (VE) can be defined as:
VE = VR + 2 V* (4)
where V* can be determined graphically from the breakthrough curve as shown in Figure 2.
This value is usually different from that of V because of the asymmetry of the breakthrough
curves.

The capacity factor of the solute, k, can be calculated from the fundamental equation of
chromatography:
VR = VM (1 + k) (5)
where VM represents the hold-up volume of the SPE cartridge. Thus,

R
M
V
k 1
V
= (6)
A very important parameter, the recovery r, can also be directly related to the breakthrough
curve. It is defined as the amount of analyte that can be recovered after the SPE procedure,
expressed in percentage of the total amount of the analyte originally present in the sample.
Knowing this quantity allows for the concentration of the analyte in the sample to be
calculated from the concentration determined in the sample extract.

The amount adsorbed in the cartridge can be denoted by ns and in the special case where the
total volume V0 is safely in excess of the equilibrium volume, ns is equal to the linear
capacity of the column, ns, lim which can be expressed by the equation [2]:
ns, lim = VM k c0 (7)
The recovery factor can now be calculated as:

s
0 0
n
r 100%
c V
= (8)
or, when possible, in the simplified expression:

M
0
V k
r 100%
V
= (9)

Direct Method

This method is very similar to the first, and allows the calculation of the fundamental
parameters using the following relations:
VR = 0.5 c0 (1)
VB = 0.05 c0 (10)

5.
VE = 0.95 c0 (11)
For the determination of the other quantities, like the number of theoretical plates, the linear
capacity and the recovery factor, the same relations used in the first method are applicable
here (3, 6, 8).

Third Derivative Method

We are going to present now in some detail an original method based upon the extreme
points of the third and first derivative curve. The disadvantage of the first two methods is
their inflexibility in the calculation of the parameters, which directly depend on the value of
c0 and dont consider the actual shape of the curve. More than often, the BC curve has an
asymmetric character, which is not taken into account by the two methods. Using
derivatives, however, emphasises the particular characteristics of each curve. In this paper,
we aim at showing that this method yields comparable results, carrying however a greater
physical significance.

A prerequisite for any of the three methods, and especially for this one, is the smoothing of
the experimental data. Due to the presence of noise and the recording of a very large number
of experimental values (around 3000 points), the data obtained presents minor fluctuations.
Although not visible in an analogue analysis, such fluctuations are a nuisance when
computer-aided interpretation is performed, especially when we are interested in the shape
of a high-order derivative, which exacerbates the irregularities.

For plotting and visualisation of data, we have used a demo version of TableCurve 2D
version 5. The program offers several smoothing algorithms, and we preferred the Savitzky-
Golay procedure which works best with high-order derivatives. This time-domain method of
smoothing is based on least squares polynomial fitting across a moving window within the
data. The method was originally designed to preserve the higher moments within time-
domain spectral peak data. The TableCurve 2D implementation of the Savitzky-Golay
algorithm offers sequential internal smoothing passes to improve overall noise reduction.
Figure 3 compares the shape of a Savitzky-Golay interpolated curve with a non-interpolated one.


Figure 3. Savitzky-Golay interpolated and non-interpolated data

Our method for the evaluation of the fundamental parameters of a breakthrough curve is
based on the following observations:
Savitzky-Golay Smoothed Data
BC curve (detail), n=200
15.5 16.5 17.5 18.5 19.5
Volume, mL
250
300
350
400
450
500
P
o
t
e
n
t
ia
l,

m
ic
r
o
V
250
300
350
400
450
500
P
o
t
e
n
t
ia
l,

m
ic
r
o
V
Savitzky-Golay Smoothed Data
BC curve (detail), n=2
15.5 16.5 17.5 18.5 19.5
Volume, mL
250
300
350
400
450
500
P
o
t
e
n
t
ia
l,

m
ic
r
o
V
250
300
350
400
450
500
P
o
t
e
n
t
ia
l,

m
ic
r
o
V

6.
the first maximum of the third derivative corresponds to the breakthrough volume;
the minimum of the third derivative (or the maximum of the first) corresponds to the
retention volume;
the second maximum of the third derivative corresponds to the equilibrium volume.

The TableCurve 2D program allows us to visualize the shape of the derivatives
superimposed on the breakthrough curve, so it is easy to compare the results obtained and
see if they have physical significance.

Using a small moving window (parameter Win n) we carefully smooth the data so that the
first region of the curve (where V0 is read) doesnt get altered by the algorithm. Usually a
value for n of 100-200 for total volumes around 130 mL is enough to obtain meaningful
results. The first maximum of the third derivative is read at this point (see Figure 4).

Figure 4. BC curve and its third derivative, for n=150, order 4 and 10 passes.
The arrow shows the first maximum of the derivative, corresponding to VB

For the retention volume, a stronger smoothing (n around 300) is required, until the first
derivative has the expected shape (Figure 5). To read the equilibrium volume, an even
stronger smoothing (values up to 800 for n) has to be applied, to get the corresponding shape
of the third derivative for large volumes (Figure 6).

At such high levels of smoothing, the first part of the interpolated breakthrough curve
presents visible alteration (although the rest of it remains pretty much unchanged), as can be
seen from Figure 7 which compares the shape of the interpolated curve for different values
of the moving window n. However, since we are no longer interested in the breakthrough
volume (which we read at the lowest possible level of smoothing assuring real significance
for the parameter), this exhibits no impediment to our estimations.
Savitzky-Golay Smoothed Data
BC curve and its 3rd derivative, n=150
0 40 80 120
Volume, mL
-7.5
-5
-2.5
0
2.5
5
7.5
10
P
o
t
e
n
t
i
a
l
,

V
0
10000
20000
30000
40000
50000
60000
70000
P
o
t
e
n
t
i
a
l
,

V
V
B

7.


Figure 5. BC curve and its first derivative, for n=250, order 4 and 10 passes.
The arrow shows the maximum of the derivative, corresponding to VR



Figure 6. BC curve and its third derivative, for n=500, order 4 and 10 passes.
The arrow shows the minimum of the derivative, corresponding to VR,
and the second maximum, corresponding to VE.
Savitzky-Golay Smoothed Data
BC curve and its 1st derivative, n=250
0 40 80 120
Volume, mL
0
250
500
750
1000
1250
1500
P
o
t
e
n
t
i
a
l
,

V
0
10000
20000
30000
40000
50000
60000
70000
P
o
t
e
n
t
i
a
l
,

V
V
R
Savitzky-Golay Smoothed Data
BC curve and its 3rd derivative, n=500
0 40 80 120
Volume, mL
-4
-3
-2
-1
0
1
2
3
P
o
t
e
n
t
i
a
l
,

V
0
10000
20000
30000
40000
50000
60000
70000
P
o
t
e
n
t
i
a
l
,

V
V
R
V
E

8.
We can also obtain the value of the retention volume from the third derivative (see Figure 6),
as the maximum of the first corresponds to the minimum of the third. But for precise results,
we recommend the first derivative, since its more simplified shape allows the positioning of
the maximum even at low levels of smoothing, compared to the third derivative. Low levels
of smoothing imply a less alteration of the primary data, hence a higher precision.

Roughly, an estimate of the relative uncertainty in the calculation of the parameters, based
on the fluctuations observed, would give a value of about 5%. A more detailed analysis
would have to be carried out in order to asses the precision of the method.


Figure 7. Savitzky-Golay interpolated curve for n = 150 and n = 500

One last observation should be made regarding the total volume of the sample V0. Since we
conducted an online experiment, there was no preset volume to be run through the cartridge,
instead we allowed for the upper plateau of the breakthrough curve to be reached (total
adsorption) and we stopped after some period of linearity. In order to asses a meaningful
value for V0 (which cannot be the volume at which the experiment was stopped, but an
earlier volume), we decided to make use of the detectors precision.

Indeed, after a time (volume) the plateau of the BC curve becomes linear, except for
background noise fluctuations. We measured the baseline noise in a separate experiment and
we decided to take as reference a value ten times greater than the background. This quantity
(in units of potential), subtracted from the maximum attained potential, gives the value
corresponding to the total volume V0.



xperimental

3.1. Summary

e have tested the behaviour of C18-bonded silica cartridges using para-chloraniline
(PCA) in different conditions of flow and analyte concentration. The parameters of
each acquired breakthrough curve were computed using all the three methods presented in
the first part of this paper. A comparison between the methods is made, as well as an
E
W
Savitzky-Golay Smoothed Data
BC curve (first region, detail), n=150
0 10 20 30
Volume, mL
-500
0
500
1000
1500
2000
P
o
t
e
n
t
ia
l,

m
ic
r
o
V
-500
0
500
1000
1500
2000
P
o
t
e
n
t
ia
l,

m
ic
r
o
V
Savitzky-Golay Smoothed Data
BC curve (first region, detail), n=500
0 10 20 30
Volume, mL
-500
0
500
1000
1500
2000
P
o
t
e
n
t
ia
l,

m
ic
r
o
V
-500
0
500
1000
1500
2000
P
o
t
e
n
t
ia
l,

m
ic
r
o
V
3.

9.
interpretation of the results for C18. Following, several new materials were tested using BC
curves, and compared to the traditional C18 stationary phase, in order to find possible new
stationary phases to be used in chromatographic and SPE applications.

3.2. Experimental Setup

ractically, one can obtain a breakthrough curve if the solution of an analyte of c0
concentration is pumped through a conditioned SPE cartridge and the effluent is
monitored online. For this, the cartridge is directly connected to the HPLC pump, and the
solute passes initially through the cartridge and then in the detector, while the signal from
the detector is acquired using a computer (Figure 8).


Figure 8. Experimental setup

An offline system has also been developed, in which the cartridge is put on a vacuum pump
ensuring the passage of the solute through the cartridge; the solute, recovered at the exit of
the cartridge, is analyzed by liquid chromatography. Using this system, it is necessary to
pass through the cartridge and to collect, in time, ratios containing a definite volume of
solute (5 mL for example). However, this method is very time-consuming and governed by a
large uncertainty concerning the sample volume.

3.3. Calibration Curves

he breakthrough curves are recorded as potential variation (in V) against time (in min).
The quantity on the abscissa can easily be changed into volume (mL) by multiplying
with the flow. From this the volume of the fittings, measured separately for our experimental
device, has to be subtracted.

In order to transform the electrical potential into units of concentration (mg/L) the following
procedure was applied. First, several samples of known concentration were injected into the
column. It was obtained thus a linear dependence between the concentration and the area of
the peak obtained by injection of the sample. Then, several breakthrough curves were
recorded at different concentrations, followed by injection of the sample. Using the previous
linear relationship, the area of the peaks obtained by injection was correlated with the
concentration. Moreover, the area can be correlated with the height of the breakthrough
curves plateau (the potential). Combining the two relationships, we obtain an equation for
the transformation of electrical potential into the corresponding concentration.



P
T
S
o
l
u
t
i
o
n

Pump UV D PC
Cartridge

10.

esults and Interpretation


or all the tabulated results, the first line correspond to the second method explained in
the theoretical part, the second line correspond to the first method and the third line
correspond to the third method (with derivative curve). We expect to have similar values,
and to test in this way the validity of the new method.

4.1. Study of C
18
-bonded silica stationary phase

Influence of analyte concentration

e have studied for different concentrations the parameters of the breakthrough curve
in order to have a reference and an order of magnitude. The results (N, k and r) for
different concentrations are supposed be the same because these parameters characterise the
couple solid phase (C18) - analyte (PCA). However, we expect an influence of the
concentration on retention volume, breakthrough and equilibrium volume.

The experimental data and the parameters of the curves are given in Table 1. Indeed, as
predicted, the values of N, k and r are stable when concentration changes. Moreover, ns (the
quantity adsorbed during the experiment) increases with the increase of concentration,
which is logical, since the same volume of sample contains a larger quantity of analyte to be
adsorbed.

We can observe that the values of the breakthrough volume obtained by the second method
(first line) are notably different and greater than the ones obtained by the other two methods.
The results given by the third derivative method are however similar to those given by the
first method.

Table 1. Influence of analyte concentration upon adsorption on C18

Conc (mg/L) VB (mL) VE (mL) VR (mL) N k n (mmol/g) r %
30.470 89.199 55.186 15.477 191.956 4.560E-04 47.724
22.519 95.395 55.186 8.044 191.956 4.560E-04 47.724 1
23.650 92.250 54.667 8.955 190.142 4.504E-04 47.277
30.320 83.495 54.362 15.948 189.077 8.562E-04 48.296
22.029 88.329 54.362 7.967 189.077 8.562E-04 48.296 2
23.140 87.360 53.700 8.852 186.762 8.409E-04 47.720
27.586 89.661 55.485 11.867 193.003 1.253E-03 50.327
16.827 96.561 55.485 5.416 193.003 1.253E-03 50.327 3
19.880 92.460 53.980 7.011 187.741 1.229E-03 48.908
25.726 84.356 51.965 11.730 180.696 1.644E-03 48.304
16.330 89.934 51.965 5.595 180.696 1.644E-03 48.304 4
17.625 85.075 51.625 6.186 179.507 1.623E-03 47.980

R
F
W
4.

11.
Influence of sample flow

We aimed to study the shape of the BC curves for different values of the flow, at 3, 4 and 5
mL/min. However, the results obtained at 3 mL/min were rather bad, due to some
experimental errors, and have to be repeated. Here are given the values obtained at flows of
4 and 5 mL/min (Table 2). As expected, we see no remarkable influence of the flow on the
intrinsic parameters of the stationary phase - analyte system (N, k and r). However, the
greater the flow, the faster the breakthrough volume is attained.

Table 2. Influence of sample flow upon adsorption on C18

Flow (mL/min) Vb (mL) Ve (mL) Vr (mL) N k ns (mmol/g) r %
35.270 92.604 58.570 20.254 203.791 4.607E-04 53.990
26.090 98.548 58.570 9.417 203.791 4.607E-04 51.370 4
28.867 94.747 53.767 14.470 186.995 4.239E-04 47.134
31.396 93.469 57.264 15.309 199.222 4.547E-04 49.516
21.228 101.046 57.264 7.004 199.222 4.547E-04 48.985 5
23.950 96.100 51.175 10.645 177.934 4.075E-04 43.790


4.2. Study of colloidal silica based materials

ew materials containing a silica-based stationary phase bonded with different
inorganic compounds have been tested for comparison with C18 and to see which one,
if any, could be a better choice for chromatographic or solid-phase extraction applications.

The results obtained are given in Table 3. The letter S in the name of the materials denotes
the addition of ferrous sulphate FeSO4 to the colloidal silica matrix, and the number
following represents the temperature at which the phase has been activated. The letter A
denotes the addition of ferrous nitrite Fe(NO3)2, while the letter M, the addition of magnetite
FeO-Fe2O3.

When we compare these results we see that the compound S is very good for an application
in SPE because k and r are rather large; however in comparison with the C18 the efficiency is
smaller. Moreover, it would be interesting to optimise this stationary phase for an
application in chromatography because the results for N are bigger than the results for other
compounds. It is also interesting to note the positive influence of temperature on the number
of plates for both materials A and M.

However, in general the compounds tested with this matrix dont behave very well because,
for example, all the capacity factors are less than 10 which is very little when compared with
a value about 200 found for the C18.





N

12.
Table 3. Results obtained for colloidal silica based materials

Material Vb (mL) Ve (mL) Vr (mL) N k ns (mmol/g) r %
1.796 5.841 3.183 17.158 5.279 2.010E-05 34.905
S_70 1.327 6.098 3.183 8.629 10.131 2.010E-05 34.905
1.500 4.733 2.733 17.493 8.557 1.709E-05 29.413
1.620 4.801 2.644 19.953 4.869 1.593E-05 35.938
S_550 1.188 5.033 2.644 9.562 8.243 1.593E-05 35.938
1.200 3.700 2.250 14.556 6.867 1.338E-05 29.912
1.695 6.584 3.159 14.306 5.084 2.163E-05 29.182
S_1000 1.170 6.626 3.159 6.918 10.045 2.163E-05 29.182
1.333 4.467 2.633 12.626 8.207 1.781E-05 23.865
1.112 8.457 3.429 5.883 2.162 2.938E-05 17.501
A_70 0.295 8.729 3.429 2.632 10.988 2.938E-05 17.501
0.567 5.567 2.900 3.705 9.140 2.598E-05 14.555
0.962 5.681 1.956 11.573 2.888 1.312E-05 21.669
A_550 0.484 5.451 1.956 4.423 5.840 1.312E-05 21.669
0.683 2.867 1.400 11.791 3.895 8.690E-06 14.452
0.879 4.734 1.737 12.358 2.269 1.126E-05 21.715
A_1000 0.499 4.492 1.737 5.074 5.073 1.126E-05 21.715
0.667 2.400 1.300 12.990 3.545 7.809E-06 15.189
0.698 4.816 1.693 8.175 4.579 1.034E-05 19.128
M_70 0.242 4.793 1.693 3.112 4.918 1.034E-05 19.128
0.400 2.533 1.197 6.085 3.184 6.709E-06 12.419
0.932 6.945 2.012 10.262 1.929 1.187E-05 18.186
M_550 0.403 5.978 2.012 3.764 6.035 1.187E-05 18.186
0.650 2.900 1.350 11.041 3.720 7.536E-06 11.210
0.923 4.667 1.806 12.686 1.439 1.064E-05 22.664
M_1000 0.523 4.556 1.806 5.118 5.316 1.064E-05 22.664
0.667 2.533 1.333 13.718 3.662 7.294E-06 15.615



4.3. Study of alcoxidic based materials

he second matrix used was an alcoxidic one improved by the addition of different
structural units at different sites. For the Mteos material was added the methyl group,
for Teos, the ethyl group and for Pteos one ethyl and one phenyl group.
When we observe the results, the Teos seems to be the best compound. For Teos at 70C the
number of theoretical plates is bigger than for other compounds, and also the recovery has a
good value, making it interesting to deepen the study of Teos as a stationary phase in
chromatography. Moreover, the values for k and r at a temperature of 550C are good
compared with the rest of materials, although not similar to the C18. This would recommend
the use of Teos 550 in SPE applications.



T

13.
Table 3. Results obtained for alcoxidic based materials

Material Vb (mL) Ve (mL) Vr (mL) N k ns (mmol/g) r %
2.204 6.262 3.479 25.308 11.164 2.169E-05 29.700
Teos_70 3.479 6.278 3.479 13.307 11.164 2.169E-05 29.700
1.875 4.875 3.025 22.723 9.577 1.872E-05 31.091
3.931 17.259 8.876 9.316 30.036 8.988E-05 44.269
Teos_550 2.636 18.381 8.876 5.247 30.036 8.988E-05 44.269
1.867 13.867 6.667 4.950 22.310 6.583E-05 32.887
2.015 10.118 4.386 9.949 14.334 3.403E-05 26.912
Teos_1000 0.889 10.627 4.386 3.776 14.334 3.403E-05 26.912
1.333 6.767 3.033 9.769 9.606 2.298E-05 18.009
0.490 5.248 1.548 5.643 4.413 8.599E-06 11.739
Pteos_70 1.548 4.975 1.548 1.678 4.413 8.599E-06 11.739
0.333 2.000 0.767 8.953 1.681 3.283E-06 5.690
2.173 15.923 5.084 8.716 16.776 4.411E-05 25.253
PTeos_550 0.663 18.129 5.084 2.995 16.776 4.411E-05 25.253
1.200 6.250 3.100 7.482 9.839 2.998E-05 14.784
0.933 7.856 2.287 8.104 6.996 1.904E-05 13.596
PTeos_1000 0.212 7.912 2.287 2.705 6.996 1.904E-05 13.596
0.600 2.650 1.325 9.869 3.633 1.009E-05 7.139
1.156 8.610 2.630 9.166 8.196 1.413E-05 21.803
Mteos_70 2.630 7.856 2.630 3.417 8.196 1.413E-05 21.803
0.900 4.000 2.200 8.071 6.692 1.136E-05 17.428
0.759 5.097 1.734 9.661 5.064 1.550E-05 17.637
MTeos_550 0.440 4.624 1.734 4.522 5.064 1.550E-05 17.637
0.533 2.400 1.267 8.574 3.429 1.042E-05 11.961
0.837 5.179 1.745 10.922 5.101 1.361E-05 16.311
MTeos_1000 0.421 4.638 1.745 4.338 5.101 1.361E-05 16.311
0.500 2.450 1.250 7.820 3.371 8.988E-06 10.778





onclusions


The purpose of this study was to test and evaluate a new method for the calculation of BC
parameters. We have seen that the proposed method gives similar values to the methods
described in literature, but also takes into account the particular shape of each curve. Using
this method, we have compared the results obtained for C18 and several other new materials,
at different conditions of concentration and flow. Thus, we have seen that the method is
generally valid and suitable for a wide application. Moreover, various conclusions can be
draw concerning the properties of the analysed materials.


C
5.

14.
References

1. A. Gelencser, G. Kiss, Z. Krivacsy, Z. Varga-Puchony, J. Chromatogr. A, 1995, 693, 217.
2. I. Liska, J. Krupcik and P.A. Leclercq, J. High Res. Chromatogr., 1989, 12, 577.