49
CHARACTERIZATION OF NITROCELLULOSES BY 2D-LC Research project 2015 Konstantina Badra Supervisor: Dr. Wim Th. Kok Master of Analytical Sciences
CHARACTERIZATION OF
NITROCELLULOSES BY 2D-LC Research project 2015
Konstantina Badra
Supervisor: Dr. Wim Th. Kok
Master of Analytical Sciences
1
Abstract
In this study an effort was made to optimize a reversed-phase method, in order to couple it
online with a size exclusion chromatography method. The SEC method would enable
collecting fractions of higher and lower molecular weight and the RP-LC method would
separate them according to their nitrogen content. It was managed to reduce the gradient
time needed to four minutes. However, the method displayed many problems, such as
breakthrough of the polymers and high backpressures. The latter occurred due to
precipitation of the polymers in the column. The column was blocked very often due to the
precipitated nitrocellulose; consequently long washing periods should be applied.
Furthermore, the SEC fractions were much diluted leading to collecting them twice. Finally,
the fractions should be spiked with water before the RP-LC analysis, in order to reduce the
polymers’ breakthrough. Due to the aforementioned the method could not be implemented
online.
2
Acknowledgements
I would like to express my sincere gratitude to professor Dr. Wim Th. Kok for the scientific
guidance that provided to me during the course of this work. I would, also like to thank him
for the patience and confidence that showed to me during this project. Without his help and
counsel the completion of this work would be immeasurably difficult. Moreover, I would like
to give special thanks to PhD candidate Maria Marioli for all the help she provided me with
when problems occurred at the laboratory work, but for her moral support, as well.
3
MSc Chemistry
Analytical Sciences
Master Thesis
Characterization of nitrocelluloses by 2D-LC
by
Konstantina Badra
August 2015
Supervisor:
Dr W. Th. Kok
Analytical Chemistry Group
van’ t Hoff Institute for Molecular Sciences
4
Table of contents I. List of figures ..................................................................................................................... 5
II. List of tables ....................................................................................................................... 7
III. Introduction ................................................................................................................... 8
1. Nitrocellulose ................................................................................................................ 8
2. Size exclusion chromatography (SEC) ............................................................................ 9
3. Reversed-phase liquid chromatography (PR-LC) ......................................................... 10
4. Evaporative light-scattering detector (ELSD) .............................................................. 11
5. Scope of the study ....................................................................................................... 11
IV. Experimental part ........................................................................................................ 11
V. Results and discussion ..................................................................................................... 12
6. ELSD detector .............................................................................................................. 12
7. Size exclusion chromatography ................................................................................... 16
a. Calibration with polystyrene samples ..................................................................... 16
b. Calculation of nitrocellulose samples molecular weight ......................................... 18
c. Collection of fractions ............................................................................................. 20
8. Reversed-phase liquid chromatography ..................................................................... 24
d. Dwell time................................................................................................................ 24
e. Breakthrough ........................................................................................................... 26
f. Developed method .................................................................................................. 35
g. Analysis of the fractions .......................................................................................... 42
VI. Conclusions and future prospects ............................................................................... 45
VII. References ................................................................................................................... 47
5
I. List of figures Figure 1. Representation of the nitrocellulose’s chemical structure. Reproduced from ref. [2]. 8 Figure 2. Representation of the chemical structure of a nitrocellulose with a nitrogen content of
12.2% and degree of substitution of 2.3 (left). Equation of the degree of substitution
estimation based on the nitrogen content and the relationship between the two (right). 9 Figure 3. Schematic representation of basic principles of SEC separation. Reproduced from ref. [10].
10 Figure 4. Schematic representation of an ELSD detector. Reproduced from ref. [18]. 11 Figure 5. RP-LC column coupled to the UV detector coupled to the ELSD detector. 12 Figure 6. SEC chromatogram of a sample dissolved in pure THF containing 13.15% (165) nitrogen
content compound for two different detector gains. 13 Figure 7. Peak areas of a sample containing both 12.20% (169) and 13.50% (177) nitrogen content
compounds dissolved in 60:40 THF:H2O (0.125 mg/mL) versus different injected amounts. 14 Figure 8. Peak heights of a sample containing both 12.20% (169) and 13.50% (177) nitrogen content
compounds dissolved in 60:40 THF:H2O (0.125 mg/mL) versus different injected amounts. 14 Figure 9. Peak areas of different concentrations’ samples containing both 12.20% (169) and 13.50%
(177) nitrogen content compounds dissolved in 60:40 THF:H2O versus different sample
concentrations. Injected volume was 4 μL. The upper equation is for the 12% compounds
whereas the lower represents the 13% compounds. 15 Figure 10. Peak heights of different concentrations’ samples containing both 12.20% (169) and
13.50% (177) nitrogen content compounds dissolved in 60:40 THF:H2O versus different sample
concentrations. Injected volume was 4 μL. The upper equation is for the 12% compounds
whereas the lower represents the 13% compounds. 15 Figure 11. SEC chromatograms of different molecular weight polystyrene samples dissolved in pure
THF (1 mg/ml). 17 Figure 12. Log (10) of the molecular mass of the highest peak from the polystyrene samples versus
the retention time of each standard. The equation shown fits the exponential line of the
calibration. 18 Figure 13. SEC chromatograms of all the samples currently in possession. 19 Figure 14. SEC chromatograms of sample 178 (13%) and its three fractions. Injection is 30 μL. 21 Figure 15. Zoom in on the SEC chromatographs of the fractions collected for the sample 178 (13%).
21 Figure 16. SEC chromatographs of sample 175 (11.10%) and its fractions. 22 Figure 17. Zoom in on the SEC chromatographs of the fractions collected for the sample 175
(11.10%). 22 Figure 18. SEC chromatogram of a mixture of nitrocellulose samples 165 (13.15%) and 171 (11.45%)
at flow rate 0.750 ml/min. 23 Figure 19. SEC chromatogram of the fractions collected from a mixture of nitrocellulose samples
165 (13.15%) and 171 (11.45%) at flow rate 1.0 ml/min. 23 Figure 20. Zoom in the SEC chromatogram of the fractions collected from a mixture of nitrocellulose
samples 165 (13.15%) and 171 (11.45%) at flow rate 1.0 ml/min. 24 Figure 21. Schematic representation of a low-pressure mixing system. Reproduced from ref. [22]. 25 Figure 22. UV detector signal for the measuring of dwell time. 25 Figure 23.Chromatogram of a sample containing 168, 169 and 176 samples dissolved in pure THF
and representation of the THF/H2O gradient applied. The concentration was 0.100 mg/ml and
the injection volume 40 μL. 26
6
Figure 24. Chromatogram of a sample containing 168, 169 and 176 samples dissolved in pure THF
and representation of the THF/H2O gradient applied. The concentration was 0.100 mg/ml and
the injection volume 40 μL. 27 Figure 25. Chromatogram of a sample containing 168, 169 and 176 samples dissolved in pure THF
and representation of the THF/H2O gradient applied. The concentration was 0.100 mg/ml and
the injection volume 40 μL. 27 Figure 26. Chromatogram of a sample containing 168, 169 and 176 samples dissolved in pure THF
and representation of the THF/H2O gradient applied. The concentration was 0.100 mg/ml and
the injection volume 40 μL. 28 Figure 27. Chromatogram of a sample dissolved in pure THF containing 11.1% (176) compound
eluted with different isocratic methods of 10, 30 and 60% THF. The concentration was 0.100
mg/ml and the injection volume 40 μL. 29 Figure 28. Chromatogram of a sample dissolved in 70:30 THF:H2O containing 12.2% (169) compound
eluted with different isocratic methods of 90, 80 70 and 60% THF. The concentration was 0.100
mg/ml and the injection volume 2.5 μL. 30 Figure 29. Blank of 70:30 THF:H2O composition under 90:10 THF:H2O isocratic conditions ran after
the analysis of sample 169 under 60:40 THF:H2O isocratic conditions. The injected volume was
2.5 μL. 30 Figure 30. Chromatograms of different amounts injected of a 12.2% nitrogen content sample (169)
dissolved in 70:30 THF:H2O (0.005 mg/mL). 32 Figure 31. Chromatograms of continuous 10 μL (500 ng injections of a 12.2% nitrogen content
sample (169) dissolved in 70:30 THF:H2O (0.005 mg/mL) and a blank injection. 32 Figure 32. Chromatograms of different amounts injected of a 12.2% nitrogen content sample (169)
dissolved in 70:30 THF:H2O (0.125 mg/mL). 33 Figure 33. Chromatograms of continuous 4 μL (500 ng injections of a 12.2% nitrogen content sample
(169) dissolved in 70:30 THF:H2O (0.125 mg/mL) and a blank injection. 34 Figure 34. Chromatograms of two different injections of a 12.20% (169) nitrogen content sample
dissolved in 60:40 THF:H2O (0.033 mg/mL). 34 Figure 35. Chromatograms of different injections of a sample containing both 12.20% (169) and
13.50% (177) nitrogen content samples dissolved in 60:40 THF:H2O (0.125 mg/mL) and one
blank injection. Injection volume is 4 μL (500 ng). 35 Figure 36. Chromatograms of two different samples. One sample containing both 12.20% (169) and
13.50% (177) nitrogen content samples and another containing 11.10% (175) nitrogen content
compound dissolved in 60:40 THF:H2O (0.125 mg/mL). Injection volume is 4 μL (500 ng). 36 Figure 37. Chromatograms of a sample containing both 12.20% (169) nitrogen content compound
dissolved in 60:40 THF:H2O (0.125 mg/mL) injected two different days. Injection volume is 4 μL
(500 ng). 37 Figure 38. Chromatograms of 4 μL blank injections dissolved in 70:30 THF:H2O before and after a 30-
minute washing period with 90:10 THF:H2O. 37 Figure 39. Reverse-phase chromatogram of a blank sample of THF:H2O 60:40. 38 Figure 40. Chromatogram of one sample containing both 12.20% (169) and 13.50% (177) nitrogen
content samples dissolved in 60:40 THF:H2O (0.125 mg/mL). Injection volume is 4 μL (500 ng).
39 Figure 41. Chromatograms of a mixture containing 169 (12.20%) and 177 (13.50%) compounds
dissolved in 60:40 THF:H2O at 0.125 mg/mL concentration. The orange and blue line represent
columns ODS-I and ODS-IIIE, respectively. Injection was 4 μL. 39 Figure 42. Chromatogram of a sample containing 176 (11.10%), 169 (12.20%) and 177 (13.50%)
compounds dissolved in THF:H2O (60:40) at 0.125 mg/mL concentration. Injected amount was
4 μL. 40 Figure 43. Chromatogram of a sample containing 176 (11.10%), 169 (12.20%) and 177 (13.50%)
compounds dissolved in THF:H2O (60:40) at 0.125 mg/mL concentration. Injected amount was
4 μL. 41
7
Figure 44. Chromatogram of a sample containing 176 (11.10%), 169 (12.20%) and 177 (13.50%)
compounds dissolved in THF:H2O (60:40) at 0.125 mg/mL concentration. Injected amount was
4 μL. 41 Figure 45. Chromatogram of a sample containing 173 (12.53%) and 168 (13.35%) compounds
dissolved in THF:H2O (60:40) at 0.125 mg/mL concentration. Injected amount was 4 μL. 42 Figure 46. Nitrogen content (%) of the nitrocellulose samples against retention time of the different
samples. The light blue, orange and grey dots represent the 11, 12 and 13% nitrogen content
samples, respectively. 42 Figure 48. Chromatograms of the three fractions of the 178 (13%) sample. 43 Figure 49. Chromatograms of the three fractions of the 169 (12.20%) sample. 43 Figure 50. Chromatograms of the two fractions of the 175 (11.10%) sample. 44 Figure 51. RPLC chromatogram of the fractions collected from a mixture of 165 (13.15%) and 171
(11.45%) samples. 44 Figure 52. RP-LC analysis of fractions collected from samples 175 (11.10%), 169 (12.20%) and 178
(13%). 45
II. List of tables Table 1. Name of the nitrocellulose’s samples and nitrogen content of each sample. 12 Table 2. SEC operating conditions. 16 Table 3. Molecular mass of the highest peak (g/mol) and retention time of the polystyrene samples
used for the calibration. 18 Table 4. Nitrocellulose samples’ name, nitrogen content (%), SEC retention time of each compound
(min), calculated molecular mass of each sample’s highest peak (g/mol) and the polydisperse
index for each sample. 20 Table 5. RP-LC operating conditions. 31
8
III. Introduction
1. Nitrocellulose Esterification of the hydroxyl groups of cellulose’s anhydroglucopyranose ring groups by
nitric acid leads to the formation of cellulose nitrate, also known as nitrocellulose (NC)
(Figure 1), which is a linear polymer built by D-glucopyranose units [1]–[3]. The reaction is
reversible and exothermic and is shown below [1].
The nitrate ester groups replace the hydroxyl groups at the ring positions 6, 2 and 3 as
depicted in Figure 1. The hydroxyl group that is replaced first is the one bonded with the
carbon in position 6 and then positions 2 and 3 follow [1], [2].
Figure 1. Representation of the nitrocellulose’s chemical structure. Reproduced from ref. [2].
NCs could be categorized according to the degree of nitration in mono-, di- and tri-nitrate
esters; the nitrogen content in each of these categories would be 10.5%, 11.1% and 14.14%,
respectively. However, the synthesis of pure mono-, di- or tri-nitrate NCs is still unachievable
or very hard to be accomplished, leading to mixtures of the three groups [2], [4]. Moreover,
the 14.14% nitrocellulose is equivalent to substitution of all the hydroxyl groups by nitro
units (Figure 2), which is practically unachievable [1]. The highest nitrogen content
nitrocellulose composed has degree of substitution equal to 3.87 [1], [5].
9
Figure 2. Representation of the chemical structure of a nitrocellulose with a nitrogen content of 12.2% and degree of substitution of 2.3 (left). Equation of the degree of substitution estimation based on the nitrogen content and the relationship between the two (right).
In this study the degree of substitution of the samples ranges from 1.99 to 2.76 for the
lowest and highest nitrogen content samples, respectively.
The nitrogen content is a factor affecting the solubility and the viscosity in organic solvents,
as well as the uses of nitrocellulose. The solubility tends to decrease with increasing nitrogen
content, whereas the viscosity increases with it [1]. Additionally, the low nitrogen content
compounds may be used for fabricating paints, inks, lacquers and photographic films, albeit
the high nitrogen content compounds could be used as dynamites and propellants [1]. For
this reason, it is of utmost importance to have a separation method which enables the
analysis of nitrocellulose compounds according to their nitrogen content. The current
method used is the Devarda’s method which includes alkaline denitration of the NC,
reduction of the resulting nitrate ions and back-titration with sulfuric acid [5]. The most
recent studies on this subject involve derivatization [6], [7] or alkaline hydrolysis [5], [8]of
the samples prior to analysis and separation by capillary electrophoresis (CE). In the former
case, D-glucopyranose derivatives are analyzed [6], [7], whereas in the latter case the
nitrogen content is calculated via quantification of nitrite and nitrate ions [5], [8]. Ion
chromatography has been used, too after alkaline hydrolysis, however CE is more cost-
effective and faster [5]. However, the aforementioned methods involve time consuming
sample preparation and it would be beneficial if it could be avoided. Another drawback of
the aforementioned methods is that they do not enable the chemical composition
distribution of the polymer samples in contrast to the RP-LC which does.
2. Size exclusion chromatography (SEC) The basic principle of SEC is the separation of macromolecules based on hydrodynamic volume differences in volume differences in solution. This difference is dependent upon the molecular mass, configuration and configuration and conformation of the molecule [9]. During a SEC analysis the macromolecules are diffused from macromolecules are diffused from the intermediate column volume into the column’s pores and vice versa as and vice versa as shown in
Figure 3 [10]. In SEC there should be no enthalpic interaction and the distribution coefficient
KSEC is derived from the following equation;
KSEC = exp(ΔS/R) = <c>i/<c>o [9] ,
Where ΔS is the conformational entropy change when a macromolecule diffuses from the
intermediate volume into the pores of the column, R the gas constant, ci is the mean solute
10
concentration within the pores of the column and co is the mean solute concentration in the
intermediate volume of the column [9].
This equation shows that the separation is based only on the macromolecule’s size taking
into account the shape and pore size of the column. Due to the fact that there are no
enthalpic interactions, isocratic elution is used [9].
It is expected in a SEC separation that the larger molecules will elute faster, since they do
not enter the pores; moreover, the smallest molecules will elute last, due to the free
diffusion in and out of the column’s pores [9], [10].
Figure 3. Schematic representation of basic principles of SEC separation. Reproduced from ref. [10].
One of SEC’s drawbacks is the use of thermonamically good solvents in order to dilute the
macromolecules (which makes the solutions non-ideal), such as tetrahydrofuran (THF) [9].
Additionally, the high flow rates used result in non-equilibrium conditions.
The SEC method allows the calculations of the polydispersity index (PDI), which is a measure
of the width of molecular weight distributions (MWD). The products of the polymerization
procedures are never homogeneous at a molecular level. The ratio of the weight average
molecular weight (Mw) to the number average molecular weight (Mn) is the polydispersity
index; PDI = Mw/Mn. The former is affiliated with the solution viscosity whereas the latter is
related to chain degradation [11]. The PDI is used to characterize the molecular weight
distribution of a polymer. Monodisperse polymers, such as polystyrene standards, which are
used for the calibration of this study, have a PDI of 1.02 to 1.10. These are the lowest values
that the PDI may take and indicate that all the polymer chains have the same length. The
higher this value becomes the broader the molecular weight distribution of the polymer is
[12]. It is important to know the PDI because it may lead to the polymerization process used
to prepare the polymer.
3. Reversed-phase liquid chromatography (PR-LC) Liquid chromatography’s basic principle is the partitioning of molecules between a mobile
and a stationary phase [10]. The mobile and stationary phase is polar and non-polar,
respectively in RP-LC. Polymers are most likely to separate by the use of gradient LC, which
encompasses solvent’s strength escalation during the analysis [10]. The separation of the
macromolecules during gradient LC is a mechanism based on precipitation-re-dissolution.
The sample is injected under such solvent conditions that the macromolecules will
precipitate in the column. A narrow band is formed at the forepart of the column by the
sample and as the thermodynamically good solvent’s percentage escalates in the mobile
phase the macromolecules begin to migrate and finally elute [10], [13]. The aforementioned
lead to the conclusion that the macromolecules’ separation is based on their chemistry. For
example, the nitrogen content of nitrocellulose which affects their solubility [1].
11
A problem that might be caused in gradient LC is the breakthrough effect. This is caused by a
high proportion of the hydrodynamically good solvent of the sample, which is sometimes
needed so that the system is not clogged. In this case a portion of the sample enters the
column with the solvent plug without being adsorbed [10], [14]. The breakthrough peak
elutes near the dead volume peak and its intensity of the breakthrough peak is affected by
several experimental parameters such as the injection volume (increases with it) and the
sample concentration (increases with it) [10], [14]. In the case of the nitrocellulose’s
separation the lower nitrogen content compounds will elute first the higher nitrogen
content compounds will follow as a result of their solubility properties.
Coupling the two aforementioned analysis techniques may lead to high separation
efficiencies and peak capacities. In addition SEC and LC show high orthogonality and may
help to extract information on both the molecular weight and the chemical composition
distribution of macromolecules [10].
4. Evaporative light-scattering detector (ELSD) The detector used in this study was concentration-sensitive detector named ELSD. Its operating principle is based operating principle is based on “nebulization of the column effluent into a stream of tiny droplets carried down a droplets carried down a short drift tube by a driving gas; the solvent is vaporized and the remaining droplets then remaining droplets then cross a narrow light beam. Measuring the scattered light on the residual droplets of the residual droplets of the non-volatile analytes leads to the signal [10], [15]–[17] (
Figure 4).
Figure 4. Schematic representation of an ELSD detector. Reproduced from ref. [18].
12
Its response may be affected by several factors, such as the solvent composition, the type of
macromolecules and their molecular weight. Furthermore, the response is not linear and
this may lead to quantification problems [10], [15]–[17]. Moreover, the sensitivity of the
detector is low and high injected amounts are demanded.
5. Scope of the study The goal of this study was to develop and optimize a two-dimensional liquid
chromatography method which would enable the nitrocellulose’s samples characterization.
The first dimension would be a size exclusion column separating the nitrocellulose according
to their size and the second would be a reversed-phase column which would separate the
SEC’s fractions depending on their nitrogen content. The combination of these two methods
might be beneficial in forensic analysis due to the fact that one would be able to find not
only the nitrogen content and molecular weight of the sample, but the origin of the
nitrocellulose, too, due to the distribution of the nitrogen.
IV. Experimental part The LC instrument used was a Waters system named Alliance Separations Module 2695. For
the SEC method, the connected to the LC was an Agilent PLgel 5 μm 105 ångström with a
molecular weight range of 60000-2000000 Da. For the RP-LC, an Eprogen 1.5 μm MICRA NPS
ODS-I 33x4.6 mm ID was coupled to the LC system. Another column that was tested was the
Eprogen 1.5 μm MICRA NPS ODS-IIIE 33x4.6 mm ID. The detectors coupled with the system
were an evaporative light scattering detector (ELSD) and a UV detector (Figure 5). The
former was a Shimadzu ELSD-LT (low temperature ELSD), whereas the latter was a Shimadzu
SPD 10Avp UV-Vis detector set at 236 and 254 nm for nitrocellulose and polystyrene,
respectively. The solvents that were used were tetrahydrofuran (THF) un-stabilized
purchased from Biosolve B.V. and deionized water 18.2 MΩ. The nitrocellulose samples were
provided by TNO Defensie en Veiligheid (TNO Defense and Security). Table 1 shows the
name and the nitrogen content of each sample supplied. For the last sample (178) there was
no information on the nitrogen content.
Table 1. Name of the nitrocellulose’s samples and nitrogen content of each sample.
Sample % N
165 13.15
168 13.35
169 12.20
171 11.45
173 12.53
175 11.10
176 11.10
177 13.50
178
13
V. Results and discussion
6. ELSD detector The ELSD detector was tested, in order to make sure that it works properly. The instruction
manual suggests that by increasing the detector’s gain by one unit the signal should be
doubled. For this reason, a sample was analyzed with the SEC method elucidated below
(Error! Reference source not found.) with the gain set at two different values.
Figure 6. SEC chromatogram of a sample dissolved in pure THF containing 13.15% (165) nitrogen content compound for two different detector gains.
Figure 6 shows the SEC chromatogram of a sample dissolved in pure THF containing 13.15%
(165) nitrogen content compound for detector gains 9 and 10. It is shown here that by
increasing the gain by one unit from 9 to 10 the peak height raises from approximately 400
14
μV to around 800 μV. This is expected as the signal of this detector should be doubled each
time the gain is escalated by one unit. This test showed that the detector worked in a proper
way regarding the signal intensities and the gain setting.
In order to test the ELSD detector’s linearity different injected amounts of the same sample
were analyzed with an RP-LC method explained thoroughly below (Table 5), and the peak
areas and heights were plotted against the injected amounts (Figure 7, Figure 8).
Figure 7. Peak areas of a sample containing both 12.20% (169) and 13.50% (177) nitrogen content compounds dissolved in 60:40 THF:H2O (0.125 mg/mL) versus different injected amounts.
Figure 8. Peak heights of a sample containing both 12.20% (169) and 13.50% (177) nitrogen content compounds dissolved in 60:40 THF:H2O (0.125 mg/mL) versus different injected amounts.
y = 0,2282x2,1065 R² = 0,9862
y = 2,6898x1,6745 R² = 0,9956
0
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
0 100 200 300 400 500 600 700
pea
k ar
ea (
μV
*sec
)
injected amount (ng)
area 12% area 13%
y = 0,022x2,0374 R² = 0,9943
y = 0,178x1,6779 R² = 0,9902
0
2000
4000
6000
8000
10000
12000
0 100 200 300 400 500 600 700
pea
k h
eigh
t (μ
V)
injected amount (ng)
height 12% height 13%
15
Figure 7 and Figure 8 show the peak areas and heights, respectively of a sample containing
both 12.20% (169) and 13.50% (177) nitrogen content compounds dissolved in 60:40
THF:H2O (0.125 mg/mL) versus different injected amounts. All the samples had a
concentration of 0.125 mg/mL and the amounts injected ranged between 125 and 625 ng. It
is shown from these figures that neither for the area nor for the height the detector is linear.
On the graphs, the equations that represent the trendlines are not linear.
Another method that was, also used to test the detector’s linearity was the injection of a
specific volume from different concentration samples. This step was conducted in order to
ascertain that the injector did not show any inconsistencies with the injection volumes and
the injections were performed accurately.
Figure 9. Peak areas of different concentrations’ samples containing both 12.20% (169) and 13.50% (177) nitrogen content compounds dissolved in 60:40 THF:H2O versus different sample concentrations. Injected volume was 4 μL. The upper equation is for the 12% compounds whereas the lower represents the 13% compounds.
y = 3E+06x1,5213 R² = 0,9571
y = 2E+06x1,2772 R² = 0,8501
0
20000
40000
60000
80000
100000
120000
140000
0 0,02 0,04 0,06 0,08 0,1 0,12 0,14
pea
k ar
ea (
μV
*sec
)
concentration (mg/mL)
12% area 13% area
16
Figure 10. Peak heights of different concentrations’ samples containing both 12.20% (169) and 13.50% (177) nitrogen content compounds dissolved in 60:40 THF:H2O versus different sample concentrations. Injected volume was 4 μL. The upper equation is for the 12% compounds whereas the lower represents the 13% compounds.
Figure 9 and Figure 10 show the peak areas and heights, respectively of different
concentrations’ samples containing both 12.20% (169) and 13.50% (177) nitrogen content
compounds dissolved in 60:40 THF:H2O versus the concentration. The concentrations ranged
from 0.033 to 0.125 mg/mL and the injected amounts fluctuated between 132-500 ng. On
the graphs, the equations that represent the trendlines are not linear. Moreover, it can be
seen that even though the same injection volume was performed, the results could not be
represented in a linear way. The graphs above confirm the fact that the ELSD detector does
not have a linear response. For this reason, quantitation is very difficult using this detector
and would be unreliable. Nevertheless, it is possible to prepare samples at a narrow
concentration range, where the response is considered linear. The limit of detection for the
method would, also be inaccurately calculated. However, it can be ascertained that below
132 ng injected, there are no observed peaks in the chromatograms.
7. Size exclusion chromatography
a. Calibration with polystyrene samples The nitrocellulose samples were analyzed via the SEC method, which conditions are shown
in Error! Reference source not found., in order to calculate their molecular mass. It is known
from the previous student conducting research with these samples that their molecular
masses fluctuate between 20000 and 700000 g/mol [19]; for this reason, polystyrene
samples of molecular masses between 19880 and 1373000 g/mol were used for the
calibration.
Table 2. SEC operating conditions.
Sample concentration (mg/mL) 1
Injection volume (μL) 30
y = 204018x1,5706 R² = 0,9594
y = 127754x1,3381 R² = 0,8553
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
0 0,02 0,04 0,06 0,08 0,1 0,12 0,14
pea
k h
eigh
t (μ
V)
concentration (mg/mL)
12% height 13% height
17
Injected amount (μg) 30
Elution Isocratic
Mobile phase composition Pure THF
Solvent composition Pure THF
Flow rate (mL/min) 1
ELSD detector gain 9
Column Agilent PLgel 5 μm 105 ångström
Temperature Ambient
Analysis time (min) 12
For the calibration with the polystyrene standards the gain of the detector was set to 5, due
to the extremely high signals obtained with higher gain values.
Figure 11. SEC chromatograms of different molecular weight polystyrene samples dissolved in pure THF (1 mg/ml).
Figure 11 shows the SEC chromatograms of the polystyrene samples. It is shown that the
lower molecular mass samples are eluted last in accordance to the theory [9], [10].
Moreover, it is noticed that as the molecular weight increases the detector’s signal
decreases, which is expected due to the fact that the ELSD detector’s signal is affected by
the molecular weight of the compounds [10], [15]–[17] . From the standards’ known
molecular masses and the SEC retention times a graph was constructed in order to calculate
the molecular masses of the nitrocellulose samples (Figure 12, Figure 13, Table 3, Error!
Reference source not found.).
18
Figure 12. Log (10) of the molecular mass of the highest peak from the polystyrene samples versus the retention time of each standard. The equation shown fits the exponential line of the calibration.
Figure 12 shows the calibration line of the polystyrene standards. The logarithmic function
of the molecular weight of the highest peak (Mp) is plotted against the retention time of
each compound. The fitting line is a linear equation in the form ax+b=0, where a is -0.7384
and b is +10.543. Table 3 shows the retention times and molecular masses of the
polystyrene samples.
Table 3. Molecular mass of the highest peak (g/mol) and retention time of the polystyrene samples used for the calibration.
Polystyrene Mp (g/mol) tR
19880 8.38
24600 8.28
43900 8.03
52400 7.94
96000 7.60
197000 7.14
775000 6.21
1373000 6.00
The values of the polystyrene standards’ molecular masses calculated by the calibration
derived equation are slightly different than the values quoted on the standards’ bottles. For
this reason a matched pair t-test was performed and there is no significant difference
between them for a 95% chance.
b. Calculation of nitrocellulose samples molecular weight Following the calibration, the nitrocellulose samples were analyzed with the SEC method
which conditions are shown in Error! Reference source not found..
y = -0,7384x + 10,543 R² = 0,9952
3,5
4
4,5
5
5,5
6
6,5
5,5 6 6,5 7 7,5 8 8,5 9
log(
10
) M
p
retention time (min)
Calibration graph
19
Figure 13. SEC chromatograms of all the samples currently in possession.
Figure 13 shows the SEC chromatograms obtained from the existing samples. It is seen that
the 11% nitrogen content samples reside in the same area of the chromatograms which
implies that they have similar molecular masses. Additionally, the rest of the samples reside
in another area of the chromatogram which indicates that they too have similar molecular
masses, which are higher than them of the 11% compounds, since they elute faster than
them. Sample 169 is lying between the two areas occupied by the other samples which
demonstrate that its molecular weight is between the highest and lowest molecular weights.
Additionally, it is noticeable that as the nitrogen content of the samples increases, their
molecular weight is escalated, too. Furthermore, it is shown here too as in Figure 11 that the
signal intensity is different among the different samples, even though the same amount was
injected. This is expected as the ELSD detector’s signal is dependent upon several factors,
such as the type of molecule or the molecular mass of the sample [10], [15]–[17]. Moreover,
the lower molecular weight samples seem to give a higher signal than the higher ones as it
happened with the polystyrene samples.
Regarding sample 178 there are no information on its nitrogen content, however analysis
with the SEC method places it in the high molecular weight samples which are the 12% and
13%. Further analysis with the RP-LC method showed it occupies the region of the 13%
samples.
20
Table 4. Nitrocellulose samples’ name, nitrogen content (%), SEC retention time of each compound (min), calculated molecular mass of each sample’s highest peak (g/mol) and the polydisperse index for each sample.
Sample % N tR (min) Mp (g/mol) PDI
165 13.15 6.73 374600 3.04
168 13.35 6.61 459384 3.07
169 12.20 7.30 142128 2.73
171 11.45 8.55 16970 1.88
173 12.53 6.85 305464 2.79
175 11.10 8.46 19776 1.85
176 11.10 8.51 18164 1.78
177 13.50 6.63 444026 3.01
178 6.70 394203 2.92
Table 4 shows the nitrocellulose samples’ nitrogen content, SEC retention time, molecular
mass of the highest peak of each compound and the polydisperse index (PDI). The latter was
calculated from the following equation; PDI = 1 + rsd2(Mw). For each time moment of the
chromatogram, the molecular weight of the peak was calculated and the relative standard
deviation of these calculations was derived. The fact that all PDIs are close to the value 2
might indicate that they were all prepared with a step polymerization process. The
molecular weights of the samples were calculated from the polystyrene calibration derived
equation as shown in Figure 12. It is shown here that the lowest molecular weights
correspond to the lower nitrogen content samples and vice versa.
c. Collection of fractions Higher and lower molecular weight samples were analyzed with SEC, in order to collect
fractions and analyze them with SEC and/or RP-LC. The samples’ concentration and the
injected amount should be high due to the fact that the collected fractions are highly
diluted. The fraction collection was facilitated by reducing the flow rate to 0.750 ml/min.
after collecting some fractions the SEC signals was too low due to the fact that they were
much diluted and consequently it was decided to collect the fractions twice for each time
frame. They were then evaporated with nitrogen and then analyzed with SEC and RP-LC.
21
Figure 14. SEC chromatograms of sample 178 (13%) and its three fractions. Injection is 30 μL.
Figure 14 show the SEC chromatograms of sample 178 (13%) and three fractions collected
from: 5.88-6.80, 6.80-7.50 and 7.50-8.31 minutes. It is shown here that even with double
fractions the peaks are so small that they are not shown compared to the SEC peak of the
178 sample. However, if zoom is applied on the chromatograms of the fractions their peaks
are shown (Figure 15Error! Reference source not found.). It is noticeable that the fractions
elute a little earlier than the large peak from the compound and this shows that probably
the concentration of the sample has an effect on the SEC separation and that the more
dilute the sample is there is a delay on its elution time. This is probably caused due to the
increase of the system’s entropy (due to the more dilute sample) and therefore this leads to
an increase to the SEC distribution coefficient and as a result the compounds are eluted
later.
Figure 15. Zoom in on the SEC chromatographs of the fractions collected for the sample 178 (13%).
0
50
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150
200
250
300
350
400
450
500
0 1 2 3 4 5 6 7 8 9 10
ELSD
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nal
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30
31
32
33
34
35
36
37
38
39
40
6 6,5 7 7,5 8 8,5
ELSD
sig
nal
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22
Figure 16. SEC chromatographs of sample 175 (11.10%) and its fractions.
Figure 16 shows the SEC chromatogram of sample 175 (11.10%) and the fractions collected
at 7.47-8.20 and 8.20-9.16 minutes. It is shown here again that a zoom in the picture is
necessary, since the peaks corresponding to the fractions are too small.
Figure 17. Zoom in on the SEC chromatographs of the fractions collected for the sample 175 (11.10%).
Figure 17 shows the SEC chromatograms of the two fractions collected from sample 175
(11.10%). It is shown here again that the fractions elute a little later than the initial samples
as was shown before. It can also be seen that in all the SEC chromatograms of the fractions
there is a peak around 9 minutes. For the higher molecular weight samples this peak did not
cause a disturbance in the chromatogram since their peaks were eluted before 9 minutes.
However, for the 11% nitrogen content samples this peak causes problems, because it co-
elutes with the fractions and overlaps with them. This peak could also exist in the initial
samples’ chromatograms as well, however their peaks are so high that it would not be
shown. Moreover, it could probably be an impurity from the system.
0
100
200
300
400
500
600
700
800
900
1000
0 1 2 3 4 5 6 7 8 9 10
ELSD
sig
nal
time (min)
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40
45
50
55
60
65
70
75
0 1 2 3 4 5 6 7 8 9 10
ELSD
sig
nal
time (min)
23
Figure 18. SEC chromatogram of a mixture of nitrocellulose samples 165 (13.15%) and 171 (11.45%) at flow rate 0.750 ml/min.
Figure 19. SEC chromatogram of the fractions collected from a mixture of nitrocellulose samples 165 (13.15%) and 171 (11.45%) at flow rate 1.0 ml/min.
Figure 18 shows the SEC chromatogram of a mixture of one high and one low nitrogen
content and molecular weight samples. Figure 19 shows the SEC chromatograms of the
fractions collected from the mixture. The peak at 9 minutes shows here, too and again the
peak height of the fractions is so low that a zoom in should be performed to see them.
0
100
200
300
400
500
600
700
800
0 2 4 6 8 10 12 14 16
ELSD
sig
nal
time (min)
20
40
60
80
100
120
140
160
180
0 2 4 6 8 10
ELSD
sig
nal
time (min)
24
Figure 20. Zoom in the SEC chromatogram of the fractions collected from a mixture of nitrocellulose samples 165 (13.15%) and 171 (11.45%) at flow rate 1.0 ml/min.
Figure 20 shows the peaks of the collected fractions from a mixture containing high and low
nitrogen content and molecular weight compounds. It is seen here again that the peak at 9
minutes overlaps with the last fraction and co-elutes with it.
8. Reversed-phase liquid chromatography For the reversed phase a non-porous column was used; MICRA NPS (non-porous silica) ODS-I
4.6x33 mm ID. This column leads to fast and efficient separations, however the efficiency is
affected by the injection volume due to the “low surface area of available for sample
interaction” [20] and it should be as low as possible [21]. Another column that was tested
was the MICRA NPS (non-porous silica) ODS-IIIE 4.6x33 mm ID which has an endcapped
monomeric C18 bonding in contrast to the MICRA NPS (non-porous silica) ODS-I 4.6x33 mm
ID which has a polymeric endcapping C18 bonding. The latter is considered to be more
stable in low and high pH environments due to better surface shielding.
d. Dwell time The dwell volume, is the system volume from the moment the mobile phase solvents are
mixed until they arrive the forepart of the column. In this study a low-pressure mixing
system was used, where the solvents are mixed before the pump. The system’s components
affecting the dwell time are the mixer, the connecting tubing, the autosampler loop as well
as the volume of the pump head [22] (Figure 21).
33
34
35
36
37
38
39
40
2 3 4 5 6 7 8 9 10 11 12
25
Figure 21. Schematic representation of a low-pressure mixing system. Reproduced from ref. [22].
The dwell time is an important factor for the analysis time, since for as long as it lasts the
compounds are eluted via isocratic conditions [23]. It could be calculated by dividing the
dwell volume to the flow rate [22], [23]. One way to counterbalance the dwell time is to
program the instrument in order to inject some minutes after the analysis has begun [24].
Unfortunately, this option was not available to the instrumentation used for this study.
Figure 22. UV detector signal for the measuring of dwell time.
Figure 22 shows the UV detector signal when the dwell time was measured. The analysis
begun with isocratic elution for 3 minutes and a gradient step at 3 minutes. The gradient
started at 70% THF and reached 95% in 6 minutes. It is shown here that the gradient starts
at 6.93 minutes and this means that the instrument has a dwell time of 3.71 minutes, which
is very close to the upper limit of the low pressure mixing systems [22]. Because the
measurement was performed with the column this gradient delay time includes the void
volume of the column, which is the space between the particles and is equal to;
-15
-10
-5
0
5
10
15
20
25
3,5 5,5 7,5 9,5 11,5 13,5 15,5
DWELL TIME
26
35%*π*r2*h
for a non-porous column, where r and h is the radius and the length of the column.
This volume was calculated to be 0.192 mL in this study which corresponds to a 38 seconds
time period when it is divided by the flow rate.
e. Breakthrough At the initial step of this study effort was made to increase the speed of the gradient method
previously conducted by Darius Boegem. This was implemented by starting the gradient
elution at a higher THF percentage compared to 50% that was used in the past. One problem
occurring then was the increased high back pressure after several injections. This was
probably the result of clogging of the column, since it was fixed after flushing with THF and
water for long times. Moreover, when a new column was used this problem stopped
appearing and the flushing time of the analysis was reduced. Furthermore, another problem
which arisen was a large breakthrough peak at the beginning of the chromatogram.
Figure 23.Chromatogram of a sample containing 168, 169 and 176 samples dissolved in pure THF and representation of the THF/H2O gradient applied. The concentration was 0.100 mg/ml and the injection volume 40 μL.
27
Figure 24. Chromatogram of a sample containing 168, 169 and 176 samples dissolved in pure THF and representation of the THF/H2O gradient applied. The concentration was 0.100 mg/ml and the injection volume 40 μL.
Figure 25. Chromatogram of a sample containing 168, 169 and 176 samples dissolved in pure THF and representation of the THF/H2O gradient applied. The concentration was 0.100 mg/ml and the injection volume 40 μL.
28
Figure 23, Figure 24 and Figure 25 show the chromatograms of the same sample containing 168, 169 and 176 compounds and different gradients ran. Figure 23 shows a gradient of 10 minutes, whereas Figure 24 and Figure 25 represent 8 and 6 minutes gradients respectively. In each case the gradient starts at 80% and reaches 95% THF. Furthermore, it is noticed in these graphs that the 11% compounds are either shown having a lower sensitivity than the higher nitrogen content compounds or not appear at all in some cases, such as in Figure 26. However, a certain conclusion could not be drawn due to the breakthrough effect that was occurring. Regarding the sensitivity it can be seen here that the higher the nitrogen content the more sensitive the detector is and the signal higher. These sensitivity differences are expected, due to the fact the ELSD detector’s signal is affected by many factors such as the molecular weight or the type of the compound [10], [15]–[17]. However, in the SEC method the low molecular weight compounds show a higher signal than the high molecular weight compounds, whereas in the RP-LC the opposite phenomenon is observed. Nevertheless, the breakthrough peak distorted the chromatograms and this was a problem that had to be solved first.
Figure 26. Chromatogram of a sample containing 168, 169 and 176 samples dissolved in pure THF and representation of the THF/H2O gradient applied. The concentration was 0.100 mg/ml and the injection volume 40 μL.
Figure 26 shows the chromatogram of the same sample as before containing 168, 169 and 176 compounds analyzed with a 4 minute gradient. It can be seen from these figures that decreasing the gradient time leads to a decrease in resolution which is something expected [10]. However, the second dimension of a two-dimensional method should be fast [25].
In order to discover what exactly the breakthrough peak was isocratic analyses of an 11%
sample were ran.
29
Figure 27. Chromatogram of a sample dissolved in pure THF containing 11.1% (176) compound eluted with different isocratic methods of 10, 30 and 60% THF. The concentration was 0.100 mg/ml and the injection volume 40 μL.
Figure 27 shows the chromatogram of an 11.1% nitrogen content sample (0.100 mg.ml)
dissolved in pure THF and eluted under different isocratic conditions of 10, 30 and 60% THF.
It is noticed from this graph that with increasing water amount in the mobile phase the
analysis time is increased. This is expected because the more viscous the mobile phase is the
slower the analysis become [25]; thus, the more the organic phase proportion at the mobile
phase the faster the analysis would be. It can, also, be seen here that with increased water
in the mobile phase the signal of the detector is decreased, which is something awaited due
to the fact that the solvent composition may have an effect on the ELSD signal [10], [15]–
[17].
Then it was decided to change the solvent’s composition, dilute the samples in 70:30
THF:H2O and half the compounds’ concentration, but this did not seem to help in reducing
the breakthrough peak. Thus, the injection volume was decreased and the samples were
dissolved in 70:30 THF:H2O. Separation under isocratic conditions was performed again in
order to determine sufficient injection volume and the gradient’s initial composition.
30
Figure 28. Chromatogram of a sample dissolved in 70:30 THF:H2O containing 12.2% (169) compound eluted with different isocratic methods of 90, 80 70 and 60% THF. The concentration was 0.100 mg/ml and the injection volume 2.5 μL.
Figure 28 shows the chromatograms of 12.2% (169) nitrogen content compound (0.05
mg/ml) dissolved in 70:30 THF:H2O, eluted under different isocratic conditions. It is noticed
here that with decreasing the injection volume and the THF amount in the mobile phase the
breakthrough peak is significantly reduced. Another observation was that under isocratic
elution of 60:40 THF:H2O the compound was not eluted. After this analysis, a blank was ran
under 90:10 THF:H2O isocratic conditions.
Figure 29. Blank of 70:30 THF:H2O composition under 90:10 THF:H2O isocratic conditions ran after the analysis of sample 169 under 60:40 THF:H2O isocratic conditions. The injected volume was 2.5 μL.
0
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180
0 1 2 3 4 5
ELSD
sig
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31
Figure 29 shows the blank of 70:30 THF:H2O composition under 90:10 THF:H2O isocratic conditions ran after the analysis of sample 169 under 60:40 THF:H2O isocratic conditions. This graph shows that the compounds were precipitated under 60% THF in the column and could not be re-dissolved and elute. Thus, when the blank was ran under 90% THF the compounds already precipitated inside the column were dissolved and eluted. This might show that when the compounds are injected in the column some time might be needed so that they precipitate. After some minutes the gradient may start and the compounds will elute. This step might not be necessary during the analysis with the instrument used for this study, due to the four minutes dwell time; however, if the method is transferred to an instrument with a lower dwell volume, the time may not be sufficient for the precipitation of the compounds. For this reason, the method was developed with a 2-minute isocratic step at the beginning of the analysis. In the following analyses the THF gradient used was the following; isocratic step of 60% THF for 2 minutes, 60-95% for 4 minutes and 95-60% for 4 minutes. Total analysis time was 10 minutes, however the 4.00 minutes dwell time is not reckoned in these 10 minutes. All the following results were obtained with this method which is shown in Table 5 unless stated otherwise.
Table 5. RP-LC operating conditions.
Sample concentration (mg/mL) 0.125
Injection volume (μL) 4
Injected amount (μg) 500
Elution Gradient
Starting mobile phase composition (THF %) 60
Starting mobile phase composition (H2O %) 40
Time of isocratic step (min) 2
Final mobile phase composition (THF %) 95
Final mobile phase composition (H2O %) 5
Time of gradient step (min) 4
Solvent composition (THF %) 60
Solvent composition (H2O %) 40
Flow rate (mL/min) 0.3
ELSD detector gain 9
Column Eprogen 1.5 μm MICRA NPS ODS-I 33x4.6 mm ID
Temperature Ambient
Total analysis time (min) 10
Different injection amounts were tested in order to discover which the maximum possible injection is without causing breakthrough.
32
Figure 30. Chromatograms of different amounts injected of a 12.2% nitrogen content sample (169) dissolved in 70:30 THF:H2O (0.005 mg/mL).
Figure 30 shows the chromatograms of a 12.2% nitrogen content sample for different
injection volumes. It can be seen here that with increasing injected amounts the
breakthrough peak enlarges. Moreover, the amount injected by the lower volumes is shown
to be too low resulting in very low peaks which are almost equal to the small breakthrough
peaks. Thus, low injected volumes of higher concentration samples should be injected
(Figure 32).
Figure 31. Chromatograms of continuous 10 μL (500 ng injections of a 12.2% nitrogen content sample (169) dissolved in 70:30 THF:H2O (0.005 mg/mL) and a blank injection.
35
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55
60
65
70
0 2 4 6 8 10 12 14 16
ELSD
sig
nal
time (min)
33
Figure 31 shows the repeatability of a 12.2% nitrogen content sample (169) dissolved in 70:30 THF:H2O (0.005 mg/mL). The amount injected was 10 μL (500 ng). It is noticeable that the method is repeatable in terms of signal intensity, peak area and height and retention time for the compound. Albeit, this does not apply to the breakthrough peak which though is repeatable regarding the retention time, its signal changes from run to run.
Figure 32. Chromatograms of different amounts injected of a 12.2% nitrogen content sample (169) dissolved in 70:30 THF:H2O (0.125 mg/mL).
Figure 32 shows the chromatograms of different amounts injected of a 12.2% nitrogen content sample (169) dissolved in 70:30 THF:H2O (0.125 mg/mL). It is shown that for a higher concentration sample, low injections result in a small breakthrough peak and a detectable signal for the compounds. It was decided then to inject 4 μL of 0.125 mg/mL samples. The repeatability of this method was then checked by consecutively injecting the same sample several times (Figure 33).
34
Figure 33. Chromatograms of continuous 4 μL (500 ng injections of a 12.2% nitrogen content sample (169) dissolved in 70:30 THF:H2O (0.125 mg/mL) and a blank injection.
Figure 33 shows the chromatograms of different 4 μL (500 ng injections of a 12.2% nitrogen content sample (169) dissolved in 70:30 THF:H2O (0.125 mg/mL) and a blank injection. It is shown here that the method is repeatable in terms of retention time, and peak area and height for the compound; however, the case is again different for the breakthrough peak, which is not repeatable in terms of peak height and area. Nonetheless, these differences are insignificant, because the peak is too small compared to the 9 times higher compound’s peak.
It was decided then to dissolve the samples in 60:40 THF:H2O in order to check if higher
volumes could be injected without showing the breakthrough effect.
Figure 34. Chromatograms of two different injections of a 12.20% (169) nitrogen content sample dissolved in 60:40 THF:H2O (0.033 mg/mL).
35
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0 2 4 6 8 10 12 14 16
ELSD
sig
nal
time (min)
35
Figure 34 shows two different injected amounts of a 12.20% nitrogen content sample
dissolved in THF:H2O at very low concentration (0.033 mg/mL). It can be seen here that a
very diluted sample in 60:40 THF:H2O does not enable higher injection volumes. It is shown
that the injection volume plays a more important role in the breakthrough effect than the
sample’s concentration. Consequently, it is more preferable to inject a low volume of a high
concentrated sample.
f. Developed method A sample of both 12.20 (169) and 13.50% (177) nitrogen content compounds was analyzed
with the developed method and is shown in Figure 35.
Figure 35. Chromatograms of different injections of a sample containing both 12.20% (169) and 13.50% (177) nitrogen content samples dissolved in 60:40 THF:H2O (0.125 mg/mL) and one blank injection. Injection volume is 4 μL (500 ng).
Figure 35 shows the chromatograms of different injections of one sample containing both
12.20% (169) and 13.50% (177) nitrogen content samples dissolved in 60:40 THF:H2O (0.125
mg/mL). It is shown that there is no baseline separation and the resolution of the two
compounds is approximately 0.8. The resolution was calculated by the following equation,
, where t1 and t2 is the retention times of the two
different compounds and w1,w2 are the widths of the peaks’ bases calculated by
extrapolating the relatively straight sides of the peaks to the baseline.
35
40
45
50
55
60
0 2 4 6 8 10 12 14 16 18 20
ELSD
sig
nal
time (min)
36
Figure 36. Chromatograms of two different samples. One sample containing both 12.20% (169) and 13.50% (177) nitrogen content samples and another containing 11.10% (175) nitrogen content compound dissolved in 60:40 THF:H2O (0.125 mg/mL). Injection volume is 4 μL (500 ng).
Figure 36 shows the chromatograms of two different samples. One sample containing both 12.20% (169) and 13.50% (177) nitrogen content samples and another containing 11.10% (175) nitrogen content compound dissolved in 60:40 THF:H2O (0.125 mg/mL). It is noticeable here that the 11% compounds overlap with the 12% compounds and are not properly separated. For this reason, a slower gradient was tested in order to have complete resolution of all the different nitrogen content compounds. Additionally, it is seen here that the low molecular weight sample shows a much smaller signal than the other compounds, in contrast to the SEC method, where the low molecular mass samples show a very high signal compared to the other compounds. A comparison, however, could not be made since the solvent composition is different in the RP-LC compared to the SEC solvent and the solvent composition might affect the ELSD detector’s signal [10], [15]–[17].
37
Figure 37. Chromatograms of a sample containing both 12.20% (169) nitrogen content compound dissolved in 60:40 THF:H2O (0.125 mg/mL) injected two different days. Injection volume is 4 μL (500 ng).
Figure 37 shows the chromatograms obtained from a sample containing a 12.20% (169)
nitrogen content compound dissolved in 60:40 THF:H2O (0.125 mg/mL) injected two
different days. It is noticeable from the graph that the method is reproducible in terms of
peak area and height, and retention time.
In adddition, an instability of the baseline is noticed in all figures from Figure 31 to Figure 37
for all the samples including the blank ones at around 10 minutes. At first the column was
rinsed with 90:10 THF:H2O for more than 30 minutes in order to ascertain that this
disturbance is not a result of compounds smeared out in the column (Figure 38).
Figure 38. Chromatograms of 4 μL blank injections dissolved in 70:30 THF:H2O before and after a 30-minute washing period with 90:10 THF:H2O.
35
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45
50
55
60
0 2 4 6 8 10 12 14 16
ELSD
sig
nal
time (min)
38
Figure 38 shows the chromatograms of 4 μL blank injections dissolved in 70:30 THF:H2O
before and after a 30-minute washing period with 90:10 THF:H2O. It is noticed that the
baseline’s instability around 10 minutes might not be compounds smeared out in the
column due to the fact that it is there after a 30-minute column wash. Another thought was
that this may be caused by the THF interacting with the PEEK tubing of the UV detector;
thus, the UV detector was disconnected and the analysis was performed with the ELSD
detector only.
Figure 39. Reverse-phase chromatogram of a blank sample of THF:H2O 60:40.
Figure 39 shows the analysis of a blank sample after the UV detector was disconnected from
the ELSD. This upward trend of the baseline when the gradient starts is still there.
Consequently it was not originating from the PEEK tubing. However, it could not be resulting
from nitrocellulose smeared out in the column, since the column used for this analysis was a
new one. Another reason for this drift could be the fact that the amount of water decreases
at that time; the ELSD detector’s temperature was set lower than 100oC and this means that
an amount of the solvent was not evaporated. When the gradient starts this amount is lower
and this could be a reason of the baseline’s drifting.
As it was mentioned at the beginning of this chapter the instrument used showed a dwell
time of almost 4 minutes. It is shown here that these 4 minutes of dwell time were enough
for the nitrocellulose to precipitate in the column without having to perform the extra 2-
minute isocratic step.
26,5
27
27,5
28
28,5
29
29,5
30
30,5
31
31,5
0 1 2 3 4 5 6 7 8
ELSD
sig
nal
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39
Figure 40. Chromatogram of one sample containing both 12.20% (169) and 13.50% (177) nitrogen content samples dissolved in 60:40 THF:H2O (0.125 mg/mL). Injection volume is 4 μL (500 ng).
Figure 40 shows the chromatogram of one sample containing a 12.20% and a 13.50% nitrogen content samples analyzed without the initial isocratic 2-minute step. The compounds are separated as before without breakthrough, however 2 minutes faster. This shows that the isocratic step is unnecessary with this instrument and that the 4 minutes dwell time are sufficient for the analysis. The peak heights are relatively repeatable among different analysis days. It is shown here that the compounds peak heights are relatively lower than in Figure 35, however the peaks are broader leading to similar values of peak areas. This could, also, be attributed to the fact that the two samples were prepared on different days and slight changes in the compound amounts might have occurred.
Another column was, also, which was the same as the one before only it was monomeric
endcapped instead of polymeric endcapped.
Figure 41. Chromatograms of a mixture containing 169 (12.20%) and 177 (13.50%) compounds dissolved in 60:40 THF:H2O at 0.125 mg/mL concentration. The orange and blue line represent columns ODS-I and ODS-IIIE, respectively. Injection was 4 μL.
20
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30
35
40
45
0 1 2 3 4 5 6 7 8 9 10
ELSD
sig
nal
time (min)
0
10
20
30
40
50
60
0 2 4 6 8 10 12 14 16
ELSD
sig
nal
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40
Figure 41 shows the chromatograms of a mixture containing 169 (12.20%) and 177 (13.50%)
compounds analyzed in the two different columns. Not many differences are observed
except for a slight (5%) decrease in the elution time. The old column which was the ODS-I
might have some precipitated nitrocellulose samples in it and consequently the compounds
might delay. On the contrary, ODS-IIIE was a new column and nothing had precipitated in it.
This could be an explanation to the shift in the gradient times; the two columns had no other
differences except for the endcapping.
Longer gradients have been ran again in order to discover if the 11% compounds could be
separated from the 12% compounds.
Figure 42. Chromatogram of a sample containing 176 (11.10%), 169 (12.20%) and 177 (13.50%) compounds dissolved in THF:H2O (60:40) at 0.125 mg/mL concentration. Injected amount was 4 μL.
Figure 42 shows the chromatogram of a sample containing 176 (11.10%), 169 (12.20%) and
177 (13.50%) compounds dissolved in THF:H2O (60:40) at 0.125 mg/mL concentration and
analyzed with a linear 10-minute gradient from 60 to 95% THF. It is shown here that even
with a slower gradient the 11% compounds do not show and they probably co-elute with the
12% compounds. This could be a proof that the 11% compounds dissolve at the same THF
percentage as the 12% compounds and they cannot be separated. The baseline’s instability
is shown with the new column, too. It is always present when the gradient starts. This leads
to the conclusion that it could not be something precipitated in the column. It could be
brittling of the PEEK tubing as the THF percentage increases.
41
Figure 43. Chromatogram of a sample containing 176 (11.10%), 169 (12.20%) and 177 (13.50%) compounds dissolved in THF:H2O (60:40) at 0.125 mg/mL concentration. Injected amount was 4 μL.
Figure 43 shows the chromatogram of a sample containing 176 (11.10%), 169 (12.20%) and
177 (13.50%) compounds dissolved in THF:H2O (60:40) at 0.125 mg/mL concentration and
analyzed with an 8-minute gradient of 60 to 95% THF. It is shown here again that the
baseline starts to increase at the moment the gradient starts and the THF percentage
increases and decreases again when the THF begins to recur at the initial proportions.
The following figures show the chromatograms of some mixtures analyzed with the RP
phase column with a 4-minute gradient.
Figure 44. Chromatogram of a sample containing 176 (11.10%), 169 (12.20%) and 177 (13.50%) compounds dissolved in THF:H2O (60:40) at 0.125 mg/mL concentration. Injected amount was 4 μL.
25
27
29
31
33
35
37
39
41
0 5 10 15 20 25
ELSD
sig
nal
time (min)
0
10
20
30
40
50
60
0 5 10 15 20 25
ELSD
sig
nal
time (min)
42
Figure 45. Chromatogram of a sample containing 173 (12.53%) and 168 (13.35%) compounds dissolved in THF:H2O (60:40) at 0.125 mg/mL concentration. Injected amount was 4 μL.
Figure 46. Nitrogen content (%) of the nitrocellulose samples against retention time of the different samples. The light blue, orange and grey dots represent the 11, 12 and 13% nitrogen content samples, respectively.
Figure 46 shows the nitrogen content of the nitrocellulose samples plotted against the
retention time of the different samples. The light blue, orange and grey dots represent the
11, 12 and 13% nitrogen content samples, respectively. It is shown here that each
percentage point occupy a specific time area with the 12 and 13% being separated and the
11 and 12% compounds eluting at the same time frame.
g. Analysis of the fractions The fractions collected from the SEC method were, also analyzed with RPLC. They were first
spiked with some amount of water so that the breakthrough effect is avoided and then
eluted on the RP column.
0
20
40
60
80
100
120
0 5 10 15 20 25
ELSD
sig
nal
time (min)
6,6
6,8
7
7,2
7,4
7,6
10,5 11 11,5 12 12,5 13 13,5 14
rete
nti
on
tim
e (m
in)
nitrogen content (%)
43
Figure 47. Chromatograms of the three fractions of the 178 (13%) sample.
Error! Reference source not found. shows the chromatograms of the three fractions
originating from 178 (13%) sample. It is shown here that all fractions are eluted at the same
time and this shows that the separation on the reversed phase is indeed based on the
nitrogen content of the samples and is independent on the molecular weight.
Figure 48. Chromatograms of the three fractions of the 169 (12.20%) sample.
Figure 48 shows the chromatograms of the three fractions originating from 169 (12.20%)
sample. It is noticeable here that one of the fractions elutes earlier than the other two. This
may indicate that the low molecular weight fractions of the same nitrocellulose sample have
a lower nitrogen content. However, this was only noticed in this sample. Another reason
why this might be caused is because of the samples’ spiking with water. It could be
attributed to a miscalculation and spiking with an amount of water that lead to a higher
proportion of H2O:THF than the usual which was 40:60. However, the third fraction still
elutes at the 12% area of the chromatogram.
20
25
30
35
40
45
0 2 4 6 8 10
ELSD
sig
nal
time (min)
23
24
25
26
27
28
29
30
31
32
33
0 1 2 3 4 5 6 7 8
44
Figure 49. Chromatograms of the two fractions of the 175 (11.10%) sample.
Figure 49 shows the reverse-phase analysis of the two fractions collected from sample 175
(11.10%). It is noticeable here that the first fraction’s peak is very small and the second
fraction’s does not exist. It can be seen from Figure 49 that the lower molecular weight
fractions show a lower signal than the higher ones. This could probably demonstrate that
the ELSD detector has a lower sensitivity towards them at this method and this is possibly a
reason of why the 11% compounds are not shown.
Figure 50. RPLC chromatogram of the fractions collected from a mixture of 165 (13.15%) and 171 (11.45%) samples.
Figure 50 shows the RP-LC chromatograms of the three fractions collected from a mixture
containing high and low nitrogen content and molecular weight. It can be seen that the 11%
compounds are not shown in the chromatogram and the 13% compounds all elute at the
same retention time. 165 sample’s peak elute earlier than the rest of the 13% compounds
25
25,5
26
26,5
27
27,5
28
28,5
29
29,5
30
2 3 4 5 6 7 8
ELSD
sig
nal
time (min)
20
22
24
26
28
30
32
34
2 3 4 5 6 7 8
ELSD
sig
nal
time (min)
45
and its retention time is the same as with a 12% compound. This could attributed to the fact
that this sample has the lowest nitrogen content comparing to the rest of the samples and is
probably dissolved in less THF than the rest of the 13% samples do.
Figure 51. RP-LC analysis of fractions collected from samples 175 (11.10%), 169 (12.20%) and 178 (13%).
Figure 51 shows the RP-LC analysis of the fractions collected from samples 175 (11.10%)
(light blue line), 169 (12.20%) (brown, grey and yellow line) and 178 (13%) (red, green and
dark blue line). It is shown here that the 12% fractions are clearly separated from the 13%
fractions. The 11% fraction, however, shows a very small peak eluted at the same time as
the 12% fractions.
VI. Conclusions and future prospects A reversed-phase method was developed in order to separate different molecular weight
nitrocellulose samples according to their nitrogen content. The gradient increases from 60 to
95% in 4 minutes. The breakthrough problem was solved by dissolving the samples in 60:40
THF:H2O and injecting a low volume. The lower molecular weight compounds (which are the
low nitrogen content compounds) are not shown in the chromatograms, probably due to co-
elution with the 12% compounds or due to lack of sensitivity of the ELSD detector.
Another problem that very often appeared was increasing high back pressure of the
instrument. In order to find its origin, the system was disconnected from the detector to the
LC. At first, it was determined that the pressure did not arose from the detector. It seemed
that the problem was caused by the initial mobile phase composition (THF:H2O 60:40) and
for this reason it was decided to check if the instrument’s mixing system was responsible for
the increased pressures. Thus, one solution with the initial mobile phase composition was
prepared, so that the LC instrument did not have to mix the solvents. However, the pressure
was still high making it impossible to run an analysis. It was concluded that the column was
blocked. This could indicate that the nitrocellulose samples were clogging the column and
that the washing of the column at the end of each analysis should be extended from 3
minutes that it was. However, something like that would increase the analysis time more
and it should thoroughly be studied, since the RP should be the second dimension of the
system and should be faster than the SEC. It could, also suggest that lower nitrocellulose
23
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31
33
35
37
39
41
5 5,5 6 6,5 7 7,5 8
ELSD
sig
nal
time (min)
46
amounts should be injected, however this seems impractical as the detection limit is not
very low. Another option would be the use of a different column capable of analyzing large
molecules, since the current column is usually implemented for small molecules, such as
epirubicin which has a molecular weight of 543.519 g/mol. Moreover the column’s loading
capacity is around 5000 ng and the injected amount in this study is 1/10 of it. If a column
with a higher loading capacity was used maybe it did not get clogged so fast, due to the
nitrocellulose precipitation, as in this case and it could last for a longer period of time. Other
solvents could be tried as well, such as ethyl acetate or butyl acetate which dissolve
nitrocellulose in order to see if the 11% compounds might get separated from the 12%
compounds. Ethyl acetate’s viscosity (0.426 cP) is slightly lower than THF’s (0.460) and this
could lead to a better mixing of the solvents, too.
This method is very difficult to be implemented online. One reason for this is that the SEC
fractions are much diluted and need to be collected twice or thrice to give signal. Another
reason is that the collected fractions should be spiked with water before they are injected to
the RP to evade the breakthrough effect. Furthermore, the RP part is still slower than the
SEC and needs more optimization regarding the column washing due to the compounds’
precipitation which eventually leads to the column’s blockage.
47
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