Bachelor Thesis Scheikunde

Characterization of nitrocellulose by 2D HPLC

Paul van Rooijen Amsterdam 16-07-2013 University of Amsterdam Analytical Chemistry Supervisors: Dr. W.Th. Kok Ing. T. Aalbers

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Samenvatting

De hoeveelheid nitraat groepen aan nitrocelluloses is van belang voor zowel de industrie als de wetenschap. Nitrocelluloses met een laag aantal nitraten wordt bijvoorbeeld gebruikt voor verf, filmmateriaal en farmacie terwijl een hoog aantal nitraten gebruikt kan worden voor explosieven. Aangezien er maar een klein verschil zit tussen laag en hoog aantal nitraten zou de mogelijkheid om dat te kunnen bepalen veel voordelen met zich meebrengen. In dit project is er geëxperimenteerd met vloeistof chromatografie om zou te proberen nitrocelluloses op verschillende kenmerken die verband hebben met de nitreringsgraad te kunnen scheiden. Nou bleek het erg lastig te zijn omdat nitrocelluloses groot en complex zijn en ook nog eens slecht oplossen. Scheiden op de verschillende polariteit eigenschappen van verschillende nitrocelluloses bleek niet mogelijk. Uit vorig onderzoek was gebleken dat nitrocelluloses verschillende oplosbaarheid vertonen als er meer of minder nitraat groepen aanwezig zijn. Daarom is er geprobeerd nitrocelluloses the scheiden op oplosbaarheid. Door nitrocelluloses eerst neer te laten slaan in een kolom werd over een periode van tijd een sterk oplosmiddel tetrahydrofuran THF geconcentreerder. Omdat bij verschillende concentraties verschillende nitrocelluloses oplossen kwamen ze op verschillende tijden uit de kolom. Aan het einde van de kolom was een UV en ELSD detector die dat konden aantonen. Met dit experiment is het gelukt om nitrocelluloses te scheiden op basis van de hoeveelheid gebonden nitraat groepen. Er bleek een lineair verband te zijn tussen de nitreringsgraad en de retentie tijd. De retentie tijd is de gepasseerde tijd tussen een injectie van een monster en de detectie daarvan na de kolom. Aan de hand van de retentie tijd zou het op deze manier mogelijk zijn de hoeveelheid nitraat groepen in nitrocelluloses te kunnen bepalen.

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Abstract

The industrial applications of nitrocellulose depend on its nitrogen content. Low nitrogen contents are used to make many daily products whereas nitrocellulose with a high nitrogen contents is used in the manufacturing of explosives and propellants. Therefore it is of importance that a method is developed to separate nitrocelluloses on different nitrogen contents. The aim of this project is to separate nitrocelluloses on size and nitrogen contents using 2D-HPLC. In earlier experiments the size-exclusion chromatography, which is the first dimension, was already successfully carried out. Therefore the second dimension of separation on nitrogen content had to be investigated. The results of these experiments show that it is possible to separate nitrocelluloses by nitrogen content using solubility as the corresponding property. When using a nonporous C18 column and mobile phase gradient consisting of THF and water a linear relationship between retention time and nitrogen content was found.

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Table of contents

Segment: Page Number: 1. Introduction 4

1.1 Short Introductory Note 4 1.2 Introduction Nitrocelluloses 4 1.3 Approach 5

2. Materials 6 2.1 Reagents and Samples 6 2.2 Instruments 7 2.3 Procedures 7

3. Results 7 3.1 Reversed Phase Gradient AcN:H2O and MeOH:H2O

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3.2 Reversed Phase Gradient AcN:THF 9 3.3 Reversed Phase Gradient THF:H2O 9 3.4 Gradient 13 3.5 Final Results 15

4. Discussion 20 5. Conclusion 21 6. Special Thanks 22 7. References 22 8. List of Abbreviations 23

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1. Introduction

1.1 Short Introductory note

The aim of this project which is provided by TNO Rijswijk is to separate nitrocelluloses based on their size and nitrogen content by the use of 2D HPLC. It was decided that the first dimension of the LCxLC would be the separation of different sized nitrocelluloses by means of size-exclusion chromatography (SEC). The second dimension will be the separation of components with different nitrogen contents by either normal or reversed phase chromatography. The size exclusion chromatography of the nitrocelluloses was already successfully carried out by Christiaan Binder and the information regarding that research can be found in his report.[1] After the SEC was operational another student Lisa Sligting tried to resolve the second dimension.[2] This report is the successor of her research and continuous on her findings. Some elements of the report of Lisa Sligting will be mentioned in this report but reading her report prior to this one is advised.[2]

1.2 Introduction Nitrocelluloses

Like the discovery of penicillin by Alexander Fleming the discovery of nitrocelluloses also was a coincidence. Christian Friedrich Schönbein was in 1846 the first man to observe the explosive properties of the nitrocelluloses after he had mopped up concentrated nitric acid with a cotton towel and left it to dry above the stove.[3] By the heat of the stove the nitrated towel ignited by itself almost without the release of smoke.[4] With this coincidence it was found that applying nitric acid on cotton, which are celluloses, form nitrocelluloses. Simply, nitrocelluloses are celluloses with substituted nitrate groups. Celluloses are linear polysaccharides that can form a chain of up to ten thousands of linked B(1-4) D-glucopyranose units.[5] On these celluloses, the carbon positions C2, C3 and C6 are bonded to hydroxyl groups which can be substituted by nitric acid in the following reaction:

Fig. 1 Nitrating reaction of cellulose by nitric acid.[2]

The nitrogen content of nitrocelluloses is of importance in many different areas. For percentages below 12.5% the nitrocelluloses can be applied as components for paints, films and varnishes whereas nitrocelluloses with percentages higher than 12.5% can be used for various explosives and propellants.[6,7] This makes not only the quantification of nitrocelluloses of importance, also the qualification as the applications depend on the nitrogen content. For example, to make high quality explosives it is best to have the highest nitrogen content possible.[8] The maximum percentage of nitrates possible (m/m) is 14.1%.[9] That is because there are only three hydroxyl groups per D-glucopyranose to be substituted by the nitrate groups. The amounts of nitrates substituted is presented as the degree of substitution (DS).[10] This degree can be calculated by the following formula:

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Fig 2. Chemical structure of a nitrocellulose with a nitrogen content of 12.2% and DS of 2.3. The equation shows the calculation of DS depending on nitrogen content.[10]

The C6 carbon has the fastest rate of substitution followed by C3 and C2 in order of reactivity. As stated before the theoretical maximum percentage of nitrogen content is 14,1% and a DS of 3. In practice however, a stable nitrocellulose with nitrogen content higher than 13.5% is not achievable.[11] The ability to qualify the nitrogen content can help to find a better synthesis of nitrocelluloses as it can help to monitor the products and thus the reaction. Because nitrocelluloses can degrade, another use of qualification the ability to monitor the degradation progress nitrocelluloses.[12] Over the past decades many analytical tools were employed to analyze nitrocelluloses. In the early stages nitrocellulose samples were analyzed by time consuming processes that took days to be successful.[14,15] Later there were analytical tools capable of quantifying nitrocellulose samples but these were not able to qualify the nitrogen contents.[13] There have been reports of qualification of nitrogen content by means of ion chromatography en capillary electrophoreses.[10,16] Also with these techniques the samples needed to be hydrolyzed or derivatized first. Qualifying nitrogen contents has still to be done without sample alteration and the need of time consuming steps. Finding a 2D HPLC analysis technique which can qualify samples of nitrocelluloses in a single step would help future analysis in regards of time. Also if samples need not to be altered like by hydrolysis, the analysis can qualify the nitrogen content of a nitrocellulose in its current state. When being able to keep samples in the original state the analysis might have a stronger position in forensics, e.g.. in cases of terrorist acts involving nitrocellulose based explosives.[17.18] If the technique will prove itself reliable it could help to identify traces of undetonated bombs which can then be used for evidence against suspects. The main question this report will try to answer is: Can nitrocelluloses be separated by the nitrogen content of the nitrocelluloses? Side question is if it is possible to do qualification analysis of nitrocelluloses. In the following paragraphs, nitrocellulose may be abbreviated by the use of NC, or in plural NCs. 1.3 Approach The plan of approach was to continue on the findings of Lisa Sligting. She tried numerous mobile phases and different columns. Her results showed that using normal or reversed phase columns yield no separation of the NCs based on the different nitrogen contents. Which is remarkable because the nitrogen content is an important parameter which affects the solubility, polarity and the viscosity of the NCs in organic solvents.[10] Still those parameters are overruled by the complexity and size of this polymer resulting in similar affinities to normal or reversed phase columns. The only separation obtained by Lisa was when using porous columns, separating the NCs on size rather than on the nitrogen content. As the second dimension of the 2D HPLC has to be solely on nitrogen content, the approach was to continue the experiments with a non-porous column. It seemed that Lisa observed that the NC sample eluted from the column at the same time as the sample solvent tetrahydrofuran (THF) in which the NCs are dissolved. What was happening was that the samples were “breaking through” with the THF resulting in no affinity to the column. In the experiments in this report, it will be attempted to eliminate the breakthrough phenomenon by using THF in the mobile phase. There are reports listing that it can be of importance to use a mobile phase which has stronger eluent

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properties than the solvents.[19] In that way the sample can have better interactions with the stationary phase. What the results of Lisa also showed is that the NCs have different solubility properties and that by changing the mobile phase NCs can deposit on the column. It seemed that NC samples with lower nitrogen content dissolve better in water than NC samples with higher nitrogen content. So the idea behind the following experiments is that changing the gradient where some NCs dissolve better in the course of time will get a separation. For this, different gradients consisting of either methanol (MeOH) or acetonitrile (AcN) in combination with water were used. If that technique will not give separations a rather unusual technique will be tried. This last technique is based upon the affinity purification techniques used for proteins, combined with the aspect of solubility. For protein affinity purification, a column is used where a selective group of proteins can bind and stick to the column, washing them off with substances that exchange or cleave the affinity bonds.[20] Instead of forming affinity bonding to the column, the plan is to let the NCs deposit on the column using high water content in the early stages of the gradient in which al NCs will deposit. Once the NCs are deposited on the column, the NCs are washed off by a gradient of an increasing THF percentage. In this way, NCs with low nitrogen content that dissolve better are removed first followed by the NCs with higher nitrogen content. This technique may prove itself resulting in separations of NCs with different nitrogen contents.

2. Materials

2.1 Reagents and Samples

The samples used for this project were supplied by TNO Rijswijk with known average nitrogen contents. Sizes of the NC samples were determined by SEC which was carried out by Christiaan Binder in the earlier stages of this project.[1] The names of the samples were based on the materials the NCs were made from. Designation of the known nitrogen contents and sizes to the different sample names can be seen in Table 1 below. The samples are dissolved in THF with a concentration of ± 5 mg mL-1. In regard of time, the first sets of experiments carried out for this report, will only make use of samples H1 (11.96%) and H2 (13.5%). This is because both samples have the lowest and highest nitrogen contents. If there is no separation possible between H1 and H2 it is most likely there also will be no separation between samples with smaller differences, H1 (11.96%) and H4 (12.71%) for example. Once a separation between samples H1 and H2 is obtained, more samples will be used. To give a wide spectrum of nitrogen contents, four samples of both the H series and NC series will be analyzed in the later stages. The complete sets are: H1, H2, H3, H4, NC2, NC11, NC53 and NC58. Al samples will be diluted to 1 mg mL-1 and injected with volumes of 10μL.

Nitrocellulose Nitrogen content Mp (g/mol) H1 11.96% 246566 H2 13.5% 391180 H3 12.15% 379327 H4 12.71% 442413 NC2 12.0-12.2% 367833 NC11 13.53% 397856 NC53 12.56% 379327 NC58 13.4-13.5% 391180 NC60 12.53% 287573 NC62 12.60% 391180 AH27 10.9-11.3% 27744 H33 11.8-12.3% 26492 Table 1. NC samples with known nitrogen contents

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2.2 Instruments

The column used in all of the experiments was a non-porous C18 column on silica support. The exact specs are:

Eprogen HPLC colum 33x4.6mm with particles sizes 1.5μm. Part No. 0446ODS101.5 Serial No. 20055F52

The HPLC setup consisted of: LC 20 AD’s pumps from Shidmadzu, UV-vis detector SPD-10AV from Shimadzu and an ELSD PL ELS 1000 from Polymerlabs. The UV-vis detector was set to detect absorption wavelengths of 237nm because on the full UV-vis spectrum the NCs absorbed best at this wavelength. The mobile phases used were analytical grade methanol, acetonitrile, tetrahydrofuran with BHT stabilizers and deionized water.

The ELSD or in full, evaporative light scattering detector, was not used in the experiments by Lisa and Christiaan. It may prove itself useful as an ELSD has an outstanding limit of detection..

For data processing Microsoft Excel 2010 was used.

2.2 Procedures

The first procedure was to create a gradient with methanol and water. Unfortunately the combination of both can result in higher viscosity and therefore then HPLC system was reaching too high backpressures. As water and acetonitrile were used by Lisa as mobile phase these two were used instead. The mixture of AcN and water created no high back pressure problem. Lisa’s report recommended to try gradients instead of an isocratic mobile phase so that was the first procedure. Gradients were made to go from a lower percentage of AcN to hundred percent over ten minutes. Starting percentages were 60% AcN, 70% AcN, 80% AcN and 90% AcN.

The second procedure was to create a gradient with AcN and THF. The mixture of these solvents gave no high back pressure problems. For this gradient, the mobile phases started at low to zero concentration of THF up one hundred percent over the course of ten minutes. As THF also absorbs at wavelength 237nm, the UV will show the gradient in the chromatographs. Because the THF is increasing along the gradient it will be harder to see the peaks of the NCs. Results of gradients with THF will be analyzed and discussed on the ELSD chromatographs rather than on the UV chromatographs.

The third procedure was to setup a gradient consisting of THF and water. For this many gradients were used and more information can be found in the segment 3.1.1.1 because setting up the gradient was based upon the results.

3. Results

3.1 Reversed Phase Gradient AcN:H2O and MeOH:H2O

As the combination of MeOH and H2O gave rise to high back pressures, over 250 bar, only a gradient of AcN:H2O was used. The following results were obtained with a gradient of increasing AcN concentration over a time of 10 minutes. Table 2 shows at which percentage of AcN the gradient started and whether the sample was breaking through with the solvent THF. Also the table provides information whether the NCs had deposition on the column. This was checked by injecting blank THF sample a couple minutes after the injection of the NC. If there was NC still in the injection loop or on the column the THF sample would remove it and show an ELSD signal.

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Nitrocellulose H1 Gradient Starting percentage AcN

(%) Breakthrough Peak with THF injected

afterwards (deposit) 100 Yes No 90 Yes Small 80 Yes Small 70 Yes Small 60 No Large 50 No Large

Nitrocellulose H2 Gradient Starting percentage AcN

(%) Breakthrough Peak with THF injected

afterwards (deposit) 100 Yes Small 90 Yes Small 80 No Large 70 No Large 60 No Large 50 No Large

Table 2. Showing the effect of different gradient starting percentages AcN on breakthrough and deposition on column

Many different gradients were performed with almost the same results. What was already mentioned in the report of Lisa Sligting was reoccurring during these gradients. After a few runs it became clear the samples were breaking through with the THF in which the samples are dissolved in, giving no differences in retention time. Depending on the starting conditions the samples either had no affinity with the column or were deposited on the column. To test if there was any NC deposited a blank THF sample was injected. Table 2 shows that the NC samples have different solubility properties as H1 still has some breaking through of the sample at a percentage of 70% AcN and a “complete” break through at a percentage of 100% AcN. The fact that H1 has a complete breakthrough at percentage of 100% AcN was based upon no signal after the injection of the THF blank. In section 3.3 there are results showing this might not be the case. For H2 there was already a small deposit of NC on the column at percentages of 90% and 100%. Below these concentrations of AcN there was no signal on the ELSD and a large ELSD signal was obtained after an injection of THF. The use of a gradient consisting of AcN and water only resulted in the rediscovery of different solubility properties depending on the nitrogen contents of the NCs. So the solubility of sample H1(11.96%) in water was better than that of H2(13.5%).

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3.2 Reversed Phase Gradient AcN:THF

By using THF in the mobile phase it was tried to resolve the problem of the samples breaking through with the solvent. As the NC samples are best dissolved in THF, a mobile phase containing THF could possibly be a better carrier mobile phase for the NCs. Many different gradients of ten minutes were tried. It seemed that changing the percentages of AcN and THF had no effect on the breaking through of the samples with the solvent THF. Using any gradient of AcN and THF did not result in separations of samples or tailing of ELSD peaks with the different NC samples. Therefore making a mobile phase with stronger dissolving properties in respect to NCs did not help.

3.3 Reversed Phase Gradient THF:H2O

For the following results a flow of 0.3ml/min was used as with 0.5ml/min the backpressure exceeded 250 bar. The problem of the high pressure was located to be at the column. The column has very small particles (1.5um) and therefore it is not unusual that it can give high backpressures. It was tried to clean the column for days using THF but with no effect. Also back flushing the column did not lower the backpressure. Although cleaning the column does not affect the high backpressure it cannot be excluded that there might be some deposit blocking the column.

First a gradient of 50% THF to 100% THF over ten minutes was run for samples H1 and H2. With this gradient the samples had different retention times on the UV and ELSD and can be seen in the chromatograms below.

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Figure 3. Chromatograph showing the UV detection signal at 237nm of sample H1 with the gradient THF:H20 (50:50 to 100:0 over ten minutes)

Figure 4. Chromatograph showing the ELSD signal of sample H1 with the gradient THF:H20 (50:50 to 100:0 over ten minutes)

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Figure 5. Chromatograph showing the UV detection at 237nm of sample H2 with the gradient THF:H20 (50:50 to 100:0 over ten minutes)

Figure 6. Chromatograph showing the ELSD detection of sample H2 with the gradient THF:H20 (50:50 to 100:0 over ten minutes)

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Figure 7. Chromatograph showing the UV detection at 237nm of blank THF sample with the gradient THF:H20 (50:50 to 100:0 over ten minutes)

Figure 8. Chromatograph showing the ELSD of blank THF ???sample H2 with the gradient THF:H20 (50:50 to 100:0 over ten minutes)

As both THF and the NCs absorb at 237 nm the UV chromatogram shows the presence of THF as well. It can be seen that the solvent THF elutes from the column around 1,40 minutes giving signal on the UV detector but not on the ELSD. To confirm this extra runs were done with either no injection or THF sample. The UV peak at 1,40 minutes was only present when a blank THF sample was injected.(Figure 7). Also when injecting THF there was no peak on the ELSD at 1,4 minutes.(Figure 8) Therefore it could be concluded there was no sample breaking through with this gradient as there was only a THF UV peak and no ELSD signal for NCs. As the percentage of THF increases in the mobile phase during the gradient the UV signal also increases and showing less significant peaks when the samples elute from the column. The ELSD chromatographs show different retention times for the samples H1 and H2. The major peaks on the ELSD correlate with the small peaks found at that time on the UV chromatographs. As for the peaks on the ELSD seem of higher resolution, future samples will be discussed on their ELSD chromatographs. Sample H1 which is the NC with the lowest nitrogen

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content has a peak at 8,6 minutes on the ELSD. Sample H2, the NC with highest nitrate content, has a peak at 9.8 minutes on the ELSD. With this gradient there is a difference of 1,2 minutes between the retention times of the samples H1 and H2. These first signs of separation were verified by repeating these conditions more than three times. These repeats showed the same retention times but the peak shapes and peak intensities were inconsistent. Both of the samples have a common small signal at around six minutes on the ELSD. It was attempted to clear the signal at six minutes by doing multiple runs without injecting and a day of washing the system with THF. Unfortunately under no circumstances would this ghost peak disappear. It was tested that the ghost peak was present with or without the injection of samples. The ghost peak could not get lower than 30 mV but it sometimes increased in intensity. When no gradients are run and the system is flushed with 50:50 THF:H20, depending on the time it did, it increases the intensity of the ghost peak for the next gradient run. Thus when running the HPLC system overnight at 50:50 THF:H2O, the first run in the morning would give ghost peak of intensities 150mV plus. The second run would already have the common ghost peak at intensities of 30 to 50mV. So whatever is causing the ghost peak to appear, most of it is not present or forming at higher percentages of THF. Luckily it seemed there was no correlation between the intensity of the ghost peak and the peaks of the samples.

3.4 Gradient

The difference of 1,2 minutes retention time between the samples had to be improved as otherwise there would be no clear separation of the remaining NC samples with nitrate contents between that of H1 and H2. For this many different gradients were run in attempt to increase the difference in retention times between the samples. It was tried to estimate the percentage of THF in which the samples would dissolve and exit the column. Unfortunately the estimation could not be implemented into other gradients as the percentage would change under different circumstances. The THF percentage of which the samples dissolve of the column are affected by the time the samples are deposited on the column and at which percentage of THF the gradients starts. For example: Starting at a THF percentage of 70%, thus a slower gradient to 100% in the same time window, some of the samples would partially breakthrough with the THF solvent at 1,4 minutes and also the retention time difference would only be increased to 2 minutes. Another variable was the rate at which the gradient increases in percentage. Rushing the gradient in short time window (1-4) minutes would result in the samples dissolving of the column earlier than expected and giving a smaller difference in retention times. Also it was discovered that gradients with a large time window would result in widening of the peaks.(figure 9)

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Figure 9. ELSD Chromatograph of a gradient of 30 minutes showing ghost peak and wide peaks of samples H1 H3 and H2 respectively.

To find the best gradient the following aspects should be kept in mind: - The NCs need to enter the column at percentage of no higher than 50% THF else there is a chance of breakthrough and the NCs not properly depositing. - The gradient has to stay below 80% THF for around five to six minutes. This makes sure the ghost peak does not interfere with the peaks of the samples and that the NCs are properly deposited. - When the NCs are properly deposited on the column, it is best to get them off the column in a shorter time window. This took more than several different gradients to set up these parameters and another lot to improve it. The following gradient seemed to give at least 3 minutes retention time difference between the samples H1 and H2:

Time(minutes) Module Action Value 0,01 Controler Start - 0,03 Pumps Total Flow 0,3 mL 0,05 Pumps Concentration THF 50% 1,00 Pumps Concentration THF 50% 5,00 Pumps Concentration THF 75%

18,00 Pumps Concentration THF 90% 19,00 Pumps Concentration THF 100% 24,00 Pumps Concentration THF 100% 25,00 Pumps Concentration THF 50% 30,00 Controler Stop -

Table 3. Gradient times and binary concentrations of THF, meaning the percentage of water in the mobile phase is hundred minus the percentage THF.

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Using this gradient resulted in the following chromatographs H1 and H2:

Figure 10. Chromatograph showing the ELSD signal of sample H1 using the gradient listed in table 3.

Figure 11. Chromatograph showing the ELSD signal of sample H2 using the gradient listed in table 3.

3.5 Final Results

The retention time of H1 was 13.7 minutes and for H2 it was 16.95 minutes. Both results were verified by multiple repeats giving the same retention times but not always same peak shape and area. Because there was a significant difference between retention times, more NC samples were run with the new gradient. The following table and graphs shows the retention times of NC samples against the nitrogen content

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Week 24 Week 26

Sample Nitration content

(%) Retention time

(min) Retention time

(min) H1 12 13,75 14

NC 2 12,1 14 14,3 H3 12,15 14,1 15

NC 53 12,5 14,3 15,75 H4 12,71 15 16

NC 58 13,45 17 17,85 H2 13,5 17,55 18

NC 11 13,53 17,7 17,9 Table 4. Retention times of samples with different nitrogen contents. Showing different retention times of the results obtained in week 24 and 26 of the year 2013.

Figure 12. Graphs showing the correlation between retention time and nitrogen content of results obtained during week 24 and 26 of the year 2013.

As both graphs show in figure 12, there is a correlation between retention time and the nitrogen content. The samples of week 24 were repeated multiple times giving the same retention times each time. So the results were repeatable during that same week. After that week the HPLC system was not used and only running slow flowrates. The week after that, week 26, the same samples were run again giving slightly longer retention times. Regardless of the lifted times, both weeks show the same relationship between nitrogen content and retention time. The following graphs show the retention time against the corresponding size Mp in g/mol instead of the nitrogen content of the samples.

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Figure 13. Graphs showing the correlation between

Sample Mp (g/mol) Nitrogen

content (%) H1 246566 12

NC 2 367833 12,1 H3 379327 12,15

NC 53 379327 12,5 NC 58 391180 13,45

H2 391180 13,5 NC 11 397856 13,53

H4 442413 12,71 Table 5. Showing relationship of the size Figure 14. Graph showing relationship of the size of of the samples with the nitrogen content the samples with the nitrogen content

Figure 13 shows that there is a slight correlation between the size and retention times for the first smaller samples and after that there is hardly a correlation visible. These graphs should show if the retention times get affected by size. If the separation of the NCS are not affected by size there should be no relationship between size and retention time. To see this effect, another graph was needed to shows the correlation between size and nitrogen content.(Figure 14) Here it can be seen that the correlation between size and retention time is almost the same as it is for the relationship between size and nitrogen content. As both the relationships look similar, the size of the NCs should have no effect on the separation.

Al the samples above were run individually and more tests were done with multiple samples in a single injection.

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Figure 15. ELSD chromatograph of samples H1 and H2 combined

Figure 16. ELSD chromatograph of samples H1 and H4 combined

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Figure 17. ELSD chromatograph of samples H2 and H4 combined

Figure 18. ELSD chromatograph of samples H1, H2 and H4 combined

These chromatographs show that it is possible to distinguish nitrocelluloses with different nitrogen contents within one sample. The resolution of the peaks is not good enough to give complete peak separations, but the retention times of the peak matches the ones of the individual samples. What also can be observed in these chromatographs is that the ghost peaks seems to be more present. The technique of letting the NC deposit on the column and letting them dissolve on different times along the gradient may have some downsides to it. The reason of this thought was that the longer this technique was used, the more the ghost peak was present. For example, in the first stages, after a night of 50% THF the ghost peak had intensities up to 200mV on the very first run in the morning. In the later stages the ghost peak had intensities up to 400mV. There is a chance that not all of injected samples exit the column during the run. In the gradient there always was a five minute period of hundred percent THF in attempt to clear out NCs that might still be stuck on the column. It is possible that after a run there still might be some NCs left on the column. Therefore more tests were conducted. The tests were done with an isocratic mobile phase of hundred percent THF with or without column, the peak areas were compared.

Peak areas in minute volts

Sample Gradient +

column Isocratic THF with

column Isocratic THF without

column H1 6834 11654 21430 H2 10184 24005 33620

Table 6. Showing the peak areas of the samples under different circumstances.

Minutes0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mV

olts

0

50

100

150

200

mV

olts

0

50

100

150

200

2488

647

6368

666

8042

627

8252

583

ELSD - Analog Board 2Nr 133 sample H1+2+4

Area

20

Figure 19. Chromatograph showing ELSD of sample H1 with isocratic THF 100% The numbers of table 6 are very clear, the peaks we see using the gradient are a lot smaller than the ones using hundred percent THF. Therefore it is possible that some NCs remain on the column. However, the results may also be explained as the result of the non-linearity of the ELSD detector. Remarkable is that even with hundred percent THF, using no column results into much larger peaks then with column. So even when using hundred percent THF, there is a possibility that some NCs get stuck on the column. This might be the reason why the same retention times can be obtained when repeating the runs but the peak shape and area are slightly random.

4. Discussion Most of the results obtained fit with the expectations and earlier findings. The results that did not meet the expectations were the second set of experiments. The second set of analyses used gradients of AcN and THF and yielded no difference in the breaking through of the samples with the solvent. It was hopeful that using THF in the mobile phase could resolve the breaking through problem. Using the gradient consisting water and acetonitrile verified the results of Lisa Sligting. It showed that samples breakthrough with the solvent or deposit on the column. It seemed that there was no effect of using a gradient instead of isocratic mobile phase containing acetonitrile and water. The final technique indeed showed that the NCs can be separated on nitrogen contents. The results of using water and THF in the mobile phase were confirming the theory of separating NCS on the solubility properties. It showed that there was a linear relationship between nitrogen content, thus solubility, and the retention time. Al of the results mentioned could be repeated and verified by doing multiple repeats. Combining samples together before injecting still resulted in the expected retention time of the individual samples. Still there are certain factors that make the results slightly differ in ghost peaks, peak shapes and peak areas. Also there is the mystery of doing the same conditions two weeks later, giving the same relationship but slightly lengthened retention times. That peak shapes and areas are not consistent is not a surprise as the nitrocelluloses are large and complex molecules and therefore may have inconsistencies. Also the nitrogen contents determined by TNO Rijswijk present the average content and not an exact single content. The peak areas

Minutes0 1 2 3 4 5 6 7 8 9 10

mV

olts

0

200

400

600

800

1000

mV

olts

0

200

400

600

800

1000

1165

4251

ELSD - Analog Board 2Nr 141 100% thf sample H1

Area

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obtained without column and isocratic THF mobile phase show that only a third of the NCs injected exit the column at the expected retention times. It is possible that low undetectable amounts exit the column later on the gradient but there is good chance that the NCs never leave the column. And it might be possible that over long period of resting in the starting conditions 50:50 THF:H2O the residues alter so that it can be flushed in the next gradient giving the ghost peak. As the last procedure was performed for a longer time the ghost peak was showing up more often and of higher intensities. It may be the reason why in the course of one week resting at starting conditions, the next week the retention times were shifted up to a minute longer. In regard to reliability of the results there are still aspects left to improve. As the relationship between nitrogen content and retention time did not change, it gives some confirmation that the results are reliable enough to make some conclusions for now. To be used in practice of qualifying NCs there are still some suggestions to be looked at first. As the column used may contain deposited or even stuck NCs, it may have had effect on the results. Therefore it is advised to try this technique again with a new, preferably low cost just in case, non-porous column. To obtain more consistent peak shapes and resolution it might help to increase the flow rate as for the last experiment a flow rate of 0.3 mL-1 was used. The idea is that using higher a flow rate means that there will a higher volume of mobile phase passing through. More volume of a certain percentage THF in which certain NCs dissolve could result in more NCs dissolving of the column in shorter period of time. The final suggestion is to analyze more different NC samples. The samples used in these experiments had a slight relation between size and nitrogen content. Because sizes are already separated in the first dimension it is important to verify the second dimension only separates on nitrogen content. Therefore it is recommended to use samples of the same size with different nitrogen contents or vice versa to verify.

5. Conclusion

As the relationship between nitrogen content and retention time stays consistent it can be concluded that nitrocelluloses can be separated using HPLC. In cases where the retention times remain constant it is also possible to qualify NCs on their nitrogen content. As there is a linear relationship between nitrogen content and the retention times, it is possible to predict the retention time of certain NCs based on their nitrogen content and vice versa. The same relationship was found between the size and nitrogen content as that of size and the retention times.(Figure 13 and 14) It implies that the size of the NCs has no effect on the retention time. To confirm this more analyses of different samples have to be made. As the peak shapes and areas are not consistent it is not possible to quantify the NCs samples using this analysis technique. The lack of consistency in peak shapes looks sloppy and therefore probably will not be sufficient for the use as evidence in court. As the nitrogen content can be predicted and qualified, this technique may prove itself useful in industries that use NCs. The results show that this analysis can distinguish low nitrated celluloses, which can be used for paint and films, from highly nitrated celluloses, which can be used for propellants and explosives. Because the samples do not have to be derivatized or hydrolyzed, the samples remain intact and thus can be used to monitor NC products of a reaction and also their degradation progress. As mentioned in the discussion, there are still some recommendations to be attempted to improve the reliability and usefulness of this technique.

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6. Acknowledgements

Here I would like to thank dr. W.Th. Kok for supervising and helping me over the course of this project. Also very special thanks to ing. T. Aalbers who helped out with the setup and all the technical problems that occured during this project. Last but not least, a thank you note for TNO Rijswijk for providing this project and the nitrocellulose samples.

7. References

[1]Binder, C. Characterization of nitrocellulose by Size-Exclusion and High Performance Liquid Chromatography; 2012 [2] Sligting, L; Characterization of nitrocellulose by 2D HPLC; 2013 [3] C.W. Saunders, L.T. Taylor, J. Energy Mater. 8, 1990, 149–203. [4] 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 [5] J.M. Talbot; Semimicor Determination of Cellulose in Biologial Materials; Uppdegraf. Anal. Biochemistry 1969 31,420-424 [6] C. Christodoulatos, T.-L. Su, A. Koutsospyros, Water Environ. Res. 73, 2001, 185–191. [7] H.F. Mark, N. Bikales, C.G. Overberger, G. Menges, J.I. Kroschwitz; Encyclopedia of Polymer Science and Engineering, second ed., Wiley-Interscience, 1985, p.139. [8] F.W. Robbins, T. Keys, The Burning Rate Behavior of Pure Nitrocellulose Propellant Samples, U.S. Army Research Laboratory, USA, 1993. [9] Selwitz C.; Cellulose Nitrate in Conservation, The Getty Conservation Institute, USA, 1988, pp. 9-14. [10] Fernández de la Ossa, M. Á.; López-López M.; Torre, M.; García-Ruiz, C. Analytical techniques in the study of highly-nitrated nitrocellulose Trends Anal. Chem. 2011, 30, 1741-1755. [11] M. Monforte, Las Polvoras y Sus Aplicaciones, UEE Explosivos, Madrid, Spain, 1992. [12] Ministry of Manpower, Singapore, Occupational safety & healthcircular, Safe Use, Handling and Storage of nitrocellulose (retrieved in September 2011, from http://www.wshc.sg/wps/themes/html/upload/cms/file/Nitrocellulose.pdf). [13] Freedman D.L.; Cashwell J.M.; Kim B.J.; Waste Manage. (Oxford, U.K.) 22, 2002, 283–292. [14] Laboratorio Químico Central de Armamento. Determinación de nitrógenonítrico y NO3, Retrieved on May 25, 2010 from http://www.enac.es/web/enac/acreditados. [15] J. Barkley, D.H. Rosenblatt; Automated nitrocellulose analysis, U.S. Army Medical Bioengineering Research and Development Laboratory, USA, Technical Report 7807, 1978. [16] Ma Ángeles Fernández de la Ossaa,b, Mercedes Torrea,b, Carmen García-Ruiza; Determination of nitrocellulose by capillary electrophoresis with laser-induced fluorescence detection. Analytica Chimica Acta. 745, 2012, 149– 155 [17] C. Cruces-Blanco, L. Gamiz-Gracia, A.M. Garcia-Campana, Trends Anal. Chem. 26, 2007, 215. [18] Bender E.C.; in A. Beveridge (Ed.), Forensic Investigations of Explosions, Taylor & Francis, London, 2003, pp. 343–388. [19] Patil N.S., Mendhe R.B., Sankar A.A., Iyer H.; Procedure for chromatography involving sample solvent with higher elution strength than the mobile phase. J Chromatogr A. 2008 Jan 11;1177(2):234-42. [20] Cuatrecasas P.; Protein Purification by Affinity Chromatography, The Journal of Biological Chemistry, Issue June 25, 1970, Vol.245, No.12

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8. List of Abbreviations

2D-LC – Two-Dimensional Liquid-Chromatography

AcN – Acentonitrile

ELSD – Evaporative Light Scattering Detector

HPLC – High Pressure Liquid Chromatography

MeOH – Methanol

NC – Nitrocellulose

NCs – Nitrocelluloses

H2O – Water

SEC – Size-exclusion Chromatography

THF – Tetrahydrofuran