Characterization of nitrocellulose by 2D HPLC

Supervisor: W. Th. Kok Amsterdam, 2013 University of Amsterdam, Analytical Chemistry Lisa Sligting

Samenvatting Nitrocellulose was ontdekt in 1845 door Christian Friedrich Schönbein, een duitse chemicus, en sinds de ontdekking werd het gebruikt voor vele toepassingen in de industrie. Nitrocellulose is een genitreerde vorm van het cellulose polymer. De nitreringsgraad van dit polymer is een belangrijke parameter, omdat het, samen met de grootte, de toepassingen van nitrocellulose bepaald. In de huidige industrie is de nitrertingsgraad van een specifiek sample eigenlijk een gemiddelde. De moleculen in een sample kunnen onderling verschillen in nitreringsgraad en grootte. Om deze reden is het belangrijk dat er een methode wordt gevonden die nitrocelluloses scheidt op deze twee eigenschappen. In dit onderzoeksproject is getracht de condities te optimaliseren voor 2D-LC. Omdat de condities voor Size-exclusion chromatografie in een eerder onderzoek al zijn geoptimaliseerd, lag de focus in dit onderzoek op het optimaliseren van de condities voor HPLC. Vier kolommen zijn getest. Drie van deze kolommen waren reversed phase kolommen. Eerst werden een C18 kolom met porie grootte van 80 Angstrom en een C18 kolom met porie grootte van 300 Angstrom getest. Beide kolommen gaven alleen een SEC effect. Verder bleek dat de nitrocellulose samples niet goed oplosbaar waren in polaire mengsels, waarin de hoeveelheid water hoger was dan 20%. Om het SEC effect tegen te gaan werden twee non-porous kolommen getest, een reversed phase en een normal phase kolom. De mobiele fases die in dit project werden geprobeerd gaven geen interactie tussen de nitrocellulose in de stationaire fase. Verder onderzoek in het scheiden van nitrocelluloses met behulp van HPLC is nodig.

Abstract Nitrocellulose was discovered in 1845 by Christian Friedrich Schönbein, a German chemist, and since then used in a wide variety of industries. Nitrocellulose is a nitrated cellulose polymer. The nitrogen content of this polymer is an important parameter. Because, together with it’s size, it determines the applications of the nitrocellulose. In the present industry the nitrogen content given for specific nitrocellulose samples is actually an average. The molecules in the sample may differ from each other in both size and nitrogen content. That is why it is important to find a separation method that can separate the nitrocellulose on both size and nitrogen content. In this research project there was attempted to optimize the conditions for 2D-LC on Nitrocellulose. Since the conditions for size-exclusion chromatography had been optimized in previous research, the focus here was on separating nitrocelluloses based on their nitrogen content with HPLC. Four columns were tried. Three of them were reversed phase columns. First a C18 column with 80 Angstrom pore size and a C18 column with 300 Angstrom as a pore size. Both of these columns were only showing SEC effects. Furthermore, the nitrocellulose samples were not soluble in polar mixtures with the water content above 20%. To get rid of the SEC effect two non-porous columns were tested, a reversed phase and a normal phase column. The mobile phases that were tried in this project for both of the non-porous columns showed no interaction between the nitrocellulose and the stationary phase. Further research on separating nitrocelluloses with HPLC is needed.

Table of Contents

1. Introduction 2. Experimental

2.1 Reagents and Samples 2.2 Instrumental 2.3 Size-Exclusion Chromatography 2.4 Reversed and Normal Phase HPLC

3. Results 3.1 SEC 3.2 Reversed Phase HPLC

3.2.1 C18 80 A and 300 A 3.2.3 Non-Porous Column

3.3 Normal Phase Non-Porous Column 4. Conclusion 5. Discussion and Recommendations References List with Abbreviations

1. Introduction

Nitrocellulose is formed by nitrating cellulose with nitric acid or another powerful nitrating agent (see fig. 1)1. Cellulose and nitrocellulose are in structure practically the same, with the only acceptation that the positions C2, C3 and C6 are, in the case of nitrocellulose occupied by nitro groups instead of hydroxyl groups. The hydroxyl group on C6 is the most favorable for nitrating, the positions C2 and C3 are equal in reactivity.

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fig. 1 nitrating reaction of cellulose

Nitrocellulose was discovered on accident by Christian Friedrich Schönbein, a German chemist. When he spilled concentrated nitric acid on his table he mopped it up with a cotton towel. He put the towel on the stove to dry. The cotton, which is almost pure cellulose had reacted with the nitric acid and had formed nitrocellulose. Because of the heat of the stove it ignited by itself. 2 Schönbein noticed that the burning of the cotton was rather fast and with hardly any smoke. That’s how he found out nitrocellulose was very suitable for explosives and smokeless gunpowder. But besides smokeless gunpowder, nitrocellulose (NC) has many other applications.

The nitrogen content is a very important parameter because not only does it have a large effect on the solubility and viscosity of the NC, it also determines the applications of nitrocellulose. The nitrogen content is given as mass percentage (m/m). Theoretically the maximum of nitrogen content, when all the hydroxyl groups are substituted by nitro groups (as the product in fig. 1), is approximately 14.1%. In the industry, however nitrocelluloses with a nitrogen content higher than 13.5% (m/m) are not synthesized.

When nitrocellulose has a nitrogen content lower than about 12% it is used in a wide variety of industries. For example, in varnishes, printing, artificial leather and pharmaceuticals. When the nitrogen content is however higher than 12-12.5% it is used in the preparation of explosives, such as dynamites, propellants and rockets.

For all these types of nitrocellulose a quality control should be customary because under storage nitrocellulose could decompose, but not only does it decompose there is another problem in the present industry; the nitrogen content given for specific NC samples is actually an average. The molecules in the sample may differ from each other in both size and nitrogen content. This is partly because there is not much known about the process of nitrating yet.

In the characterization and determination of nitrocellulose many different techniques have been used. The most common technique in determining the nitrogen content of nitrocellulose is ion chromatography, but the aim of this project was to find an adequate method to determine both the size and the nitrogen content of NCs.3-6, 8

The final objective of this project is to investigate if there are large differences in size and nitrogen content within one sample. The idea is to use two-dimensional liquid chromatography (2D-

LC). NC Samples could be separated depending on their size by size exclusion chromatography (SEC) and by high pressure liquid chromatography (HPLC). By separating them on size and nitrogen content it would be possible to find out if there are differences in the properties of nitrocelluloses within one sample. The present industry delivers nitrocellulose with an average size and nitrogen content, but as described relatively small differences in nitrogen content mean very large differences in applications. So using NCs with an average nitrogen content could be a risk. And also, finding out more about the factors that influence the process of nitrating cellulose, would be economically advantageous.

In previous research, one has found a good separation method with SEC for various NC samples.

The aim of this project was to find a way to separate NC samples on their nitrogen content by reversed and/or normal phase liquid chromatography. This is the second step to 2D-LC.

2. Experimental

The aim of this research was to find a way to perform 2D-LC so nitrocellulose samples could be separated based on both their size as well as their nitrogen content. In order to do so two steps had to be taken. First, the right conditions for size exclusion chromatography had to be found. Second, there had to be found a way to separate NCs with HPLC based on their nitrogen content.The first step in 2D-LC was taken in earlier research. SEC was performed in an earlier research project.7 The results turned out successful and will be presented in chapter 3 of this report. For the HPLC there could be taken advance from the fact the nitrogen content has an effect on the solubility of the NC. The higher the nitrogencontent the more apolair it will be. Taken this into account different mobile phases and columns could be tried.

2.1 Reagents and Samples

TNO Rijswijk supplied twelve Nitrocellulose samples. An average nitrogen content for each sample is known. The samples are shown in table 1. The samples were already dissolved in tetrahydrofuran (THF) (± 5 mg mL-1), in order to transport them. The different names have something to do with the starting material of each sample. As was mentioned before, the size of each sample was already determined in earlier research with SEC and results will be presented in this report as well in chapter 3. Nitrocellulose Nitrogen content H1 11.96% H2 13.5% H3 12.15% H4 12.71% NC2 12.0-12.2% NC11 13.53% NC53 12.56% NC58 13.4-13.5% NC60 12.53% NC62 12.60% AH27 10.9-11.3% H33 11.8-12.3% Table 1. NC samples supplied by TNO Rijswijk, with the average nitrogen content for each sample given. To give an easy-view, only four of these NC samples will be shown for most of the results. Given, H3, NC11, NC60 and AH27, which cover the whole range of nitrogen content percentages.

For determining the sizes of each NC sample polystyrene standards were used. The Mp and

Mw/Mn values of these standards are shown in table 2.

Mp Mw/Mn

6950 1.03 19850 1.02 70950 1.03 126700 1.03 197300 1.02 299400 1.02 523000 1.03 735500 1.02 1112000 1.03

Table 2. Polystyrene standards

For SEC THF was chosen as the mobile phase. Because all the NC samples dissolved well in THF and it has a relatively low UV cut off range, which is practical for the UV measurements.

For HPLC different mobile phases have been tried: Acetonitirle (AcN), Methanol (MeOH),

Acetone and mixtures with water (H2O). 2.2 Instrumental

All standards and samples were analyzed with a Shimadzu UV-Vis detector (SOD-10 AV, one wavelength used) equipped with a pump from Waters (2690 Alliance) and an auto sampler. Data processing was achieved with software from Empower.

The size exclusion column was a Plgel 5 𝜇𝑚 105 Å column. For the reversed and normal phase chromatography various columns were used; A Supelco Discovery C18 column (150 mm x 4.6 mm, 5 𝜇𝑚 particle diameter), a C18 wide pore (100 mm x 4.6 mm), a NPS SILICA column (33 mm x 4.6 mm, 1.5 𝜇𝑚) and an ODS I NPS column (33 mm x 4.6 mm, 1.5 𝜇𝑚) for the normal phase HPLC.

For the final data treatment Microsoft excel version 2007 and 2010 was employed. 2.3 Size-exclusion chromatography For the SEC experiments samples were injected with a concentration of approximately 1 mg mL-1. 2.4 Reversed and normal phase HPLC For the HPLC samples with a concentration of approximately 1.2 mg mL-1 were injected. For most experiments a blank was injected as well. The blank was pure THF, because the NC samples were dissolved in THF.

The challenge for HPLC was to avoid the SEC effect, therefore various columns have been used.

3. Results

All the runs were done with UV-Vis detection at 236 nm.

3.1 SEC

The first technique that was used is SEC. The optimized conditions were already determined in previous research. To not waste any sample, polystyrene standards were used first because they are comparable in size to the NCs. The SEC measurements from previously research were repeated to check the samples that had been in storage for over five months.

The measurements were significantly the same as fig. 2 shows.

(a)

(b) Fig. 2. Chromatograms of all NC samples (a) stored for 5 months; (b) newly prepared. Experimental conditions: flow rate, 1 mL min-1; mobile phase, THF.; inj. vol.,10 µL; NC samples dissolved in THF

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The size of each sample was determined with the polystyrene standards. The sizes are shown in table 3. Nitrocellulose Mp H1 246566 H2 391180 H3 379327 H4 442413 NC2 367833 NC11 397856 NC53 379327 NC58 391180 NC60 287573 NC62 391180 AH27 27744 H33 26492 Table 3. The sizes of the NC samples, determined with the polysterene standards. Although the size may not have been effected significantly by the period of storage, the nitrogen content of the samples could be effected because NCs can decompose under storage. So fore the HPLC new samples were supplied by TNO Rijswijk. Once again the NCs were dissolved in THF with concentrations of approximately 5 mg mL-1.

3.2 Reversed Phase HPLC

The second step to 2D-LC was separating different NC samples based on their nitrogen content by HPLC. Since the hydrophobic character of nitrocellulose increases with increase of the nitrogen content, reversed phase HPLC seemed like a suitable technique. Three different reversed phase columns were tried. Starting with two C18 columns with different pore sizes and followed by a non-porous column.

3.2.1 C18 80 Å and 300 Å

The first column that was tried in this experiment was the C18 80 Å column. Figure 3 shows the chromatogram for 100% AcN as the mobile phase. The blanc is THF, as the samples are dissolved in THF.

Fig. 3. Chromatograms of 4 NCs in THF, blank: THF. Experimental conditions: flow rate, 1 mL min-1; mobile phase, 100% AcN; inj. vol. 50 𝜇𝐿.

As can been seen, the THF peak comes at a retention time of approximately 1.6 minutes, while the peaks for the NCs come earlier. This indicates that there is only a SEC separation.

After trying some other mobile phases, 80 Angstrom seemed to be to small of a pore size for the bulky nitrocellulose molecules and there was decided to use a column with a pore size of 300 Angstrom. Figure 4 shows the chromatogram for 100% AcN as the mobile phase.

In these chromatograms only H3 and NC60 are shown, which are with a nitrogen content of respectively 12.15 and 12.53% somewhere in the middle of the range of NC samples. In figure 5 the chromatograms of the 80 Ångström and the 300 Ångström are compared and as can been seen, the pattern of peaks looks the same. Although the peaks of the 300 Ångström column are lower (because the injection volume was smaller), the only relevant difference is that the retention time of the 80 Ångström is larger than for the 300 Ångström column, meaning that there still is the so called SEC effect.

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Fig. 2. Chromatograms of 4 NCs in THF, blank: THF. Experimental conditions: flow rate, 1 mL min-1; mobile phase, 100% AcN; inj. vol., 10 𝜇𝐿.

Fig. 5. Chromatograms of 2 NCs in THF for the 80 A en 300 A columns, blank: THF. Experimental conditions: flow rate, 1 mL min-1; mobile phase, 100% AcN; inj. vol., 50 𝜇𝐿 for 80 A, 10 𝜇𝐿 for 300 A.

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There were other mobile phases tried. First, making the mobile phase step by step more polar with water. Figures 6-9 show the chromatograms for the different mobile phases.

Fig. 6. Chromatograms of 2 NCs in THF, blank: THF. Experimental conditions: flow rate, 1 mL min-1; mobile phase, 20:80 H2O:AcN; inj. vol., 10 𝜇𝐿.

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Fig. 7. Chromatograms of 2 NCs in THF, blank: THF. Experimental conditions: flow rate, 1 mL min-1; mobile phase, 30:70 H2O:AcN; inj. vol., 10 𝜇𝐿.

Fig. 8. Chromatograms of 2 NCs in THF, blank: THF. Experimental conditions: flow rate, 1 mL min-1; mobile phase, 40:60 H2O:AcN; inj. vol., 10 𝜇𝐿.

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Fig. 9. Chromatograms of 2 NCs in THF, blank: THF. Experimental conditions: flow rate, 1 mL min-1; mobile phase, 50:50 H2O:AcN; inj. vol., 10 𝜇𝐿.

At first glance the patterns of the chromatograms look the same, as for the mobile phase of 100% AcN, but when taken a closer look there are two trends slightly striking out. First there is a small peak appearing after the THF peak, this indicates that the NCs are starting to show some reversed phase effect. Second there is the peak for the blank appearing at the nitrocellulose peak. This is the most flagrant for the chromatogram with the 40:60 H2O:AcN as a mobile phase. The problem here might be that the NCs were not completely dissolving in the more polar mobile phases so some of it participated in the injector, than when a new blank was injected the NC dissolved again and therefore it looks like the blank is containing NC.

To verify this suggestion the same runs were repeated, but this time injecting the blank three times in a row. Figure 10 shows the chromatogram for the mobile phase of 40:60 (H2O:AcN) that ran right after the 30:70 mobile phase. As can be seen in this chromatogram the peak that appeared at the nitrocellulose peak, almost completely disappeared with the third blank, due to the fact that all the NC that participated in the injector had been dissolved in the first and second blanks.

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Fig. 10. Chromatogram of the blank injected three times in a row after 30:70 H2O:AcN runs were finished, blank: THF. Experimental conditions: flow rate, 1 mL min-1; mobile phase, 40:60 H2O:AcN; inj. vol., 10 𝜇𝐿.

Because the Nitrocelluloses seemed to be partly precipitating in H2O:AcN mixtures there

was taken a closer look at the solubility of the NCs in these mixtures. Table 4 shows the results.

H2O:AcN H3 (12.15%) NC60 (12.53%)

100% AcN Good Good 20:80 Good Good 30:70 Good Poor 40:60 Poor Bad 50:50 Bad Bad 40:60 Bad Bad

Table 4. Solubility of samples H3 and NC60 in water:acetonitirle mixtures (with given nitrogen content).

AS the table shows, a mixture of 30:70 water:AcN is already starting to cause problems with the solubility of NC60. And as the polarity increases the solubility decreases. So these mixture were unsuitable as a mobile phase. Another mobile phase that could be tried was methanol. The solubility of the NCs was checked for 100% MeOH en mixtures with water and is shown in table 5.

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H2O:MeOH AH27 (10.9-11.3%) NC60 (12.52%) NC11 (13.53%)

100% MeOH Good Good Good 10:90 Good Good Poor 20:80 Good Poor Bad 30:70 Poor Poor Bad Table 5. Solubility of samples AH27, NC60 and NC11 in water:methanol mixtures (with given nitrogen content). As expected NC11, with the highest nitrogen content is giving the most trouble with dissolving in water mixtures. The first three mobile phases of table 3 were tried and the chromatograms are shown in figure 10-12. Because NC11 is hardly dissolving it wasn’t injected for the water mixtures.

Fig. 10. Chromatograms of 4 NCs in THF, blank: THF. Experimental conditions: flow rate, 1 mL min-1; mobile phase, 100% MeOH; inj. vol., 10 𝜇𝐿

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Fig. 11. Chromatograms of 3 NCs in THF, blank: THF. Experimental conditions: flow rate, 1 mL min-1; mobile phase, 10:90 H2O:MeOH; inj. vol., 10 𝜇𝐿

Fig. 12. Chromatograms of 3 NCs in THF, blank: THF. Experimental conditions: flow rate, 1 mL min-1; mobile phase, 20:80 H2O:MeOH; inj. vol., 10 𝜇𝐿

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From the chromatograms it becomes clear that there is still only a SEC effect. To get rid of this effect there was decided to use a wide-pore column. The results will be discussed in the next paragraph.

3.2.2 Non-Porous Column

For the non-porous column there was decided to start with 100% AcN as a mobile phase as well. In figure 13 the chromatogram is shown.

Fig. 13. Chromatograms of 4 NCs in THF, blank: THF. Experimental conditions: flow rate, 1 mL min-1; mobile phase, 100% AcN; inj. vol., 10 𝜇𝐿 As the chromatogram shows the SEC effect has disappeared. The nitrocellulose peaks are now coming at the same time as the THF peak, meaning that there is no interaction between the analyte and the stationary phase of the column at all. The retention time of the peaks is approximately 0.45 minutes. When calculating the minimal retention of this column by using the size of the column (33 mm x 4.6 mm ID) and the flow rate (1 mL min-1), a retention time of 0.5 minutes is found. This is without taking the void volume into account, so correcting for the void volume should give a retention time of approximately 0.45 minutes. To try to retrieve interactions between the column and the NCs other mobile phases were tried. Figure 14 and 15 show the chromatograms of 10:90 and 20:80 H2O:AcN mixtures. For the 20:80 mixture the flow rate had to be decreased because the pressure got to high (>270 bar), that is why the retention of the peaks has increased.

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Fig. 14. Chromatograms of 4 NCs in THF, blank: THF. Experimental conditions: flow rate, 1 mL min-1; mobile phase, 10:90 water:AcN; inj. vol., 10 𝜇𝐿

Fig. 15. Chromatograms of 4 NCs in THF, blank: THF. Experimental conditions: flow rate, 0.5 mL min-1; mobile phase, 20:80 H2O:AcN; inj. vol., 10 𝜇𝐿

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Because the H2O:AcN mixtures are not giving any interaction MeOH and H2O:MeOH mixtures were tried. The results are shown in figures 16-18.

Fig. 16. Chromatograms of 4 NCs in THF, blank: THF. Experimental conditions: flow rate, 1 mL min-1; mobile phase, 100% MeOH; inj. vol., 10 𝜇𝐿

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Fig. 17. Chromatograms of 4 NCs in THF, blank: THF. Experimental conditions: flow rate, 1 mL min-1; mobile phase, 10:90 H2O:MeOH; inj. vol., 10 𝜇𝐿

Fig. 18. Chromatograms of 4 NCs in THF, blank: THF. Experimental conditions: flow rate, 1 mL min-1; mobile phase, 20:80 H2O:MeOH; inj. vol., 10 𝜇𝐿

As the chromatograms show, there is still no interaction between the analyte and the column for any of the mobile phases tried. Making the mobile phase more polar should normally increase the retention of (apolar) analyte, but in this case that wasn’t an option because the NCs are not dissolving well in those mixtures. Finally there was decided to use a normal phase column. Because solubility in polar solvents seemed to be one of the problems for the NCs a normal phase column could be the solution. The results are discussed in the next paragraph.

3.3 Normal Phase Non-Porous Column

For this normal phase column, the same mobile phases as for the reversed phase non-

porous column were tried. The chromatograms are shown in figures 19-24. The only exception is the 20:80 H2O:AcN mixture. For the normal phase column the pressure did not get to high so the flow rate could be kept at 1 mL min-1. Besides this insignificant difference the chromatograms of the normal phase column look exactly the same as for the reversed phase column. Given that the size of the column is the same there was concluded that there was no interaction with the stationary phase for this column neither.

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Fig. 19. Chromatograms of 4 NCs in THF, blank: THF. Experimental conditions: flow rate, 1 mL min-1; mobile phase, 100 AcN; inj. vol., 10 𝜇𝐿

Fig. 20. Chromatograms of 4 NCs in THF, blank: THF. Experimental conditions: flow rate, 1 mL min-1; mobile phase, 10:90 H2O:AcN; inj. vol., 10 𝜇𝐿

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Fig. 21. Chromatograms of 4 NCs in THF, blank: THF. Experimental conditions: flow rate, 1 mL min-1; mobile phase, 20:80 H2O:AcN; inj. vol., 10 𝜇𝐿

Fig. 22. Chromatograms of 4 NCs in THF, blank: THF. Experimental conditions: flow rate, 1 mL min-1; mobile phase, 100% MeOH; inj. vol., 10 𝜇𝐿

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Fig. 23. Chromatograms of 4 NCs in THF, blank: THF. Experimental conditions: flow rate, 1 mL min-1; mobile phase, 10:90 H2O:MeOH; inj. vol., 10 𝜇𝐿

Fig. 24. Chromatograms of 4 NCs in THF, blank: THF. Experimental conditions: flow rate, 1 mL min-1; mobile phase, 20:80 H2O:MeOH; inj. vol., 10 Typ hier uw vergelijking.

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BlancoH3NC11NC60AH27

4. Conclusions

In this project there was attempted to find a method for separating nitrocelluloses on both their size as well as their nitrogen content. The method presented was 2D-LC. The conditions for size-exclusion chromatography had been optimized in previous research. The aim of this project was to find a way to separate NCs based on their nitrogen content with HPLC. This would be the second step to 2D-LC. The first columns tried, C18 columns with pore sizes of 80 and 300 Angstrom, were only showing SEC effects. Besides, there was discovered that the NC samples weren’t dissolving well in mobile phases were the water percentage was above 20. NC samples with higher nitrogen content were giving more trouble with dissolving in the more polar phases. So the nitrogen content has a large effect on the solubility. To get rid of the SEC effect two non-porous columns were tried, a reversed phase and a normal phase column. Both of these columns showed no interaction for the mobile phases that have been tried.

5. Discussion and recommendations

In this research project there were only so many mobile phases that could be tried. Polar

solvents are causing trouble with the solubility of the NCs, but maybe other mobile phases are worth trying. Besides trying other mobile phases, trying other columns can be an option for further research as well. In fact, this would probably be a more reasonable option. Over the last 15 years the opportunities in the preparation of monolithic capillary columns have increased tremendously.7 Therefore monolithic columns seem like suitable options for further research on separating NCs with HPLC.

Furthermore there is the option of a gradient. In this research project all the experiments were isocratic, meaning that the mobile phase composition remained constant throughout the procedure. Trying a gradient, making the mobile phase from polar to slightly more apolar could give the aimed separation.

References

1. Picture made with ChemDraw 2. Epsom and Ewell History Explorer:Christian Friedrich Schönbein (18 October 1799 - 29

August 1868). Retrieved January 20, 2013, from: http://www.epsomandewellhistoryexplorer.org.uk/Schonbein.html

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4. Quye, A.; Littlejohn, D.; Pethrick, R. A.; Stewart, R. A. Accelerated aging to study the degradation of cellulose nitrate museum artefacts Polym. Degrad. Stab. 2011, 96, 1934-1939

5. Eeltink, S.; Hilder, E. F.; Geiser, L.; Svec, F.; Fréchet J. M. J.; Rozing, G. P.; Schoenmakers P. J.; Kok, W. Th. Controlling the surface chemistry and chromatographic properties of methacrylate-ester-based monolithic capillary columns via photografting J. Sep. Sci. 2007, 30, 407-413

6. López-López M.; Alegre, J. M. R.; García-Ruiz, C.; Torre, M. Determination of the nitrogen content of nitrocellulose from smokeless gunpowders and collodions by alkaline hydrolysis and ion chromatography Anal. Chim. Acta. 2011, 2, 196-203

7. Binder, C. Characterization of nitrocellulose by Size-Exclusion and High Performance Liquid Chromatography 2012

8. Lloy, J.B.F. Detection and Differentiation of NC Traces of Forensic Science Interest with Reductive Mode Electrochemical Detection at a Pendent Mercury Drop Electrode Coupled with Size-Exclusion Chromatography Anal. Chem. 1984, 56, 1907-1912

9. Christodoulatos, C.; SU, T.-L.; Koutsospyros, A. Kinetics of the Alkaline Hydrolysis of Nitrocellulose Water Environ. Res. 2001, 73, 185-19

List with abbreviations NC(s) Nitrocellulose(s) 2D-LC Two-Dimensional Liquid-Chromatography SEC Size-Exclusion Chromatography HPLC High Pressure Liquid Chromatography THF Tetrahydrofuran AcN Acetonitrile MeOH Methanol H2O Water A Angstrom