Polymer: Hewa Othman Ghareeb, Wolfgang Radke
Polymer: Hewa Othman Ghareeb, Wolfgang Radke
Polymer: Hewa Othman Ghareeb, Wolfgang Radke
Polymer
journal homepage: www.elsevier.com/locate/polymer
a r t i c l e i n f o a b s t r a c t
Article history: A liquid chromatographic method was developed allowing separating cellulose acetates (CA) with
Received 1 December 2012 respect to degree of substitution (DS). The normal-phase gradient separation, which is based on an
Received in revised form adsorptionedesorption mechanism, uses a multi-step gradient of dichloromethane (DCM) and methanol
20 March 2013
(MeOH) on a bare silica column as stationary phase. Applicability of the developed multi-step gradient
Accepted 21 March 2013
allows separating CAs within the DS-range DS ¼ 1.5e2.9, which is the target DS-range for most CA
Available online 28 March 2013
applications. To the best of our knowledge the developed method for the first time allows determination
of the DS-distribution of intact CA chains over a wide DS-range.
Keywords:
Cellulose acetate
Ó 2013 Elsevier Ltd. All rights reserved.
Degree of substitution
Degree of substitution distribution
0032-3861/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.polymer.2013.03.041
H.O. Ghareeb, W. Radke / Polymer 54 (2013) 2632e2638 2633
composed of at least eight different monomer units, which are the 2. Experimental section
differently substituted AGUs. Today, it is hardly possible to separate
intact cellulose chains of such complexity by the fractions of 2.1. Materials
the differently substituted AGUs. In our somehow more naïve pic-
ture we regard cellulose derivatives as being composed of only CA samples with average DS ranging from DS ¼ 1.5 to DS ¼ 2.9
two kind of structural units, the AGUs and the substituents. A were used. Five of the samples (sample 4, DS ¼ 1.72; sample 13,
separation with regard to DS is therefore similar to a separation of DS ¼ 2.42; sample 14, DS ¼ 2.45; sample 15, DS ¼ 2.45; sample 17,
binary copolymers according to chemical composition. Thus, the DS ¼ 2.92) were kindly provided by Rhodia Acetow GmbH (Freiburg
DS-distribution is the analogue to the chemical composition dis- im Breisgau, Germany). The others were prepared in our laboratory
tribution of binary copolymers. The efforts on separation and by partial saponification of a commercial high DS sample (sample
characterization of cellulose derivatives, particularly CAs, with 16, DS ¼ 2.60, Acetati, Italy). Information on the average DS, weight
respect to the DS-distribution are very limited and some of the average molar masses (Mw), weight average degrees of polymeri-
attempts described in literature are briefly discussed here: zation (DPw) and molar mass dispersity (Ðm) of the samples are
SEC with MALLS/RI/UV detections has been used to gain insight summarized in Table 1. The details on the synthesis and charac-
on the substitution distribution along the chains of CA [38]. The terization of the samples are given elsewhere [48]. Dichloro-
procedure requires the presence of substituents that can be methane (DCM), dimethyl acetamide (DMAc), dimethyl sulphoxide
detected by an UV-detector in relation to the molar mass deter- (DMSO) and methanol (MeOH) (VWR, Darmstadt, Germany) were
mined by MALLS/RI. It was found that DS varies with molar mass, of HPLC grade and used as received.
with the lower molar mass fractions being less substituted than the
higher molar mass fractions. However, the DS gradient over the 2.2. Chromatographic system and conditions
MMD curve does not represent a true separation in terms of DS,
because SEC separation is based on size, not on chemical compo- For the chromatographic separations a Shimadzu HPLC system
sition. Furthermore, since CAs do not contain UV-active groups, consisting of a DGU-14A degasser, an FCV-10ALvp solvent mixing
they had to be modified to the respective amide derivatives. Thus, chamber, an LC-10ADvp pump and an SIL 10ADvp auto sampler
the modification procedures have to be quantitatively performed in were used. For detection an evaporative light scattering detector
order to achieve reliable results. (ELSD, model PL-ELS 1000, Polymer Laboratories, UK) was added.
Other techniques used to obtain insight into the CCD of CAs are The detector was operated at a nebulization temperature of 50 C,
fractionated precipitation and thin layer chromatography (TLC). an evaporation temperature of 90 C and a gas flow of 1.0 SLM. The
Fractionated precipitation involves dissolving a solid polymer in a flow rate of the mobile phase was 1.0 mL/min unless mentioned
good solvent and precipitating the desired fractions stepwise by otherwise. Data collection and processing were performed using
decreasing the solubility as a result of the controlled addition of a ‘WinGPC Software version 7.0’ (Polymer Standards Service GmbH,
non-solvent. However, this method is laborious and requires large Mainz, Germany).
amounts of solvents and samples. In addition it is not very selective The experiments were performed on a pure silica stationary phase
and difficult to automate. In most cases, the isolated fractions of CAs Nucleosil, 5 mm particle size, 100
A pore diameter, 250 mm 4.0 mm
varied in DP but were of the same DS [39e43]. Thus, the separa- I.D. (Macherey & Nagel GmbH, Düren, Germany). The column tem-
tions were based on molar mass rather than on chemical compo- perature was kept constant at 35 C using a column oven K4 (Techlab
sition. Kamide et al. evaluated the use of TLC for the separation of GmbH, Erkerode, Germany). The injected sample volume was 15e
CAs according to DS and molar mass [44,45]. By stepwise changing 20 mL with concentrations of 1.5e2.0 g/L, if not stated otherwise.
the eluent composition, they were able to identify experimental
conditions where clear dependences of retardation factor (Rf) on
either DS or molar mass were observed. The resulting separations Table 1
Characterization data of samples used (samples from the industrial source are
according to DS and molar mass were found to be independent of
marked in grey).
molar mass and DS, respectively. The methods allowed calculating
both the DS-distribution and the MMD. However, TLC is difficult to Sample name DSa LS-Mwb,e [g/mol] DPwc Ðmd
quantify and has a poor reproducibility. Sample 1 1.53 57,600 254 3.0
Regarding the application of gradient chromatography, two sep- Sample 2 1.59 72,700 318 3.4
Sample 3 1.66 73,500 317 3.5
aration systems for different DS-ranges are reported in literature. Sample 4 1.72 44,600 190 2.6
Both systems followed reversed-phase liquid chromatography under Sample 5 1.81 68,100 286 3.2
different conditions. The first system reported by Floyd et al. allowed Sample 6 1.87 64,900 269 3.4
the separation of cellulose diacetate in the range of DS ¼ 2.3e2.7 Sample 7 1.92 63,200 260 3.2
Sample 8 1.95 76,400 313 3.5
[46]. The separation was carried out on a poly(styrene-co-divinyl
Sample 9 2.09 70,700 283 3.3
benzene) based column in a linear gradient from acetone/water/ Sample 10 2.16 71,900 284 3.2
MeOH (4:3:1) to acetone in 15 min at a flow rate of 0.8 mL/min. The Sample 11 2.19 71,000 279 3.5
samples eluted in the order of increasing average DS. The second Sample 12 2.27 74,400 289 3.3
system reported by Asai et al. was applied for the separation of cel- Sample 13 2.42 64,400 244 3.2
Sample 14 2.45 64,700 244 3.3
lulose triacetate in the range of DS ¼ 2.7e2.9 [47]. The separation was Sample 15 2.45 93,300 352 3.4
performed on a Waters Novapak-phenyl column in a gradient from Sample 16 2.60 69,800 257 3.5
chloroformeMeOH (9/1):MeOHewater (8/1) [2:8] to 100% in 28 min Sample 17 2.92 175,000 613 4.4
at a flow rate of 0.7 mL/min. a
Determined by 1H NMR [48].
Both systems allow calculating DS-distributions from a linear b
Determined by SECeMALLS in N,N-dimethyl acetamide/lithium chloride [48].
c
correlation between DS and retention volume. However, the appli- Calculated from Mw and DS [48].
d
cability of both methods is confined to the DS-ranges investigated. Based on PMMA-equivalent molar mass [48].
e
Note: The molar masses of chemically heterogeneous copolymers have to be
Therefore, the aim of the present paper was to develop a chro- regarded as apparent molar masses. For the present samples, the error due to
matographic method capable of separating CAs according to DS chemical heterogeneity was estimated to be of approximately the same size as the
over a wide DS-range. experimental error in LS measurement.
2634 H.O. Ghareeb, W. Radke / Polymer 54 (2013) 2632e2638
DMSO was used as a sample solvent. The eluents were DCM as a weak 0.1 mg/mL of the lowest and highest DS CA (sample 1, DS ¼ 1.53 and
eluent and MeOH as a displacer. sample 17, DS ¼ 2.92, respectively) were prepared in DMSO/DCM
The void volume (V0 ¼ 2.8 mL) of the column was determined (10/90, v/v) and MeOH was added dropwise. It was found that the
from the elution volume of a low molar mass polystyrene standard samples were soluble until the MeOH content reaches 62% for the
(Mw of PS ¼ 410 g/mol) using pure tetrahydrofuran (THF) as eluent. sample of highest and 80% for the lowest DS.
The dwell volume (Vd) was determined to be 2.3 mL by subtracting A simple linear 10 min gradient running from 100% DCM to 100%
the void volume from the onset of the increasing UV-signal due to a MeOH was applied to confirm that CAs (dissolved in DMSO) adsorb
linear gradient starting from pure THF and running to THF con- completely from DCM as initial eluent on the silica stationary
taining 0.3% acetone. phase, while the addition of MeOH causes desorption. The resulting
normalized chromatograms along with the eluent composition at
3. Results and discussion the detector are shown for selected samples (sample 1, DS ¼ 1.53;
sample 5, DS ¼ 1.81; sample 6, DS ¼ 1.87 and sample 17, DS ¼ 2.92)
For developing the separation method, a bare silica stationary in Fig. 1a.
phase was used. As can be seen from Fig. 1a, all the peaks elute within the gradient
In a previous study only DMSO and DMAc/LiCl were identified to (i.e. at times larger than 5.1 min) but at different elution times, i.e. at
dissolve all our samples, irrespective of DS [48]. Since the samples different eluent compositions. This means that all the samples are
were only partially soluble in DMAc without LiCl, DMSO was initially adsorbed onto the stationary phase despite the use of the
preferred as solvent due to the incompatibility of non-volatile LiCl DMSO as sample solvent. Desorption of the low DS samples in the
with evaporative light scattering detection (ELSD). However, DMSO DS-range DS ¼ 1.5e1.8 occurs at significantly higher MeOH contents
was not selected as the initial eluent since it creates a high back than required to elute samples of DS > 1.8. Samples of the DS > 1.8
pressure and detector noise. Most importantly, however, DMSO acts are not separated but coelute. For all samples except for sample 17
as desorption promoting liquid (desorli) and thus the use of DMSO as (DS ¼ 2.92) a sharp peak appears at a low elution volume within the
eluent did not result in polymer adsorption to the stationary phase. gradient prior to the elution of the main broad sample peak.
Seeking for conditions resulting in polymer adsorption, 0.1 mL of To increase the resolution for the high DS samples the gradient
1.0 mg/mL solutions of the lowest and highest DS CA (sample 1, slope was systematically reduced by running linear 10 min gradi-
DS ¼ 1.53 and sample 17, DS ¼ 2.92, respectively) in DMSO were taken ents from 100% DCM to 50%, 35% and 13% MeOH, respectively. The
and a second solvent (in this case, DCM) was added dropwise to identify normalized chromatograms of selected samples are shown in
the composition at which precipitation occurs. It turned out that the Fig. 1bed. As the gradients becomes more shallow, the peaks of the
DMSO solutions could be infinitely diluted with DCM without visible higher DS samples shift to higher elution volumes and a better
precipitation, despite the fact that the samples could not be dissolved resolution for samples of higher DS is obtained. However, bimodal
directly in pure DCM. Therefore, dissolution of the CAs in DMSO and chromatograms were also observed for the shallow gradients. The
subsequent dilution with DCM appeared to be a suitable way to inject origin of these bimodalities will be investigated in further experi-
the samples at adsorbing conditions without precipitation. ments below.
Methanol is known to be a strong displacer in normal phase As can be seen from Fig. 1a, samples of low DS require up to
chromatography, but is a non-solvent for CA. To identify the approximately 50% MeOH for complete elution of the peak. Thus,
maximum amount of methanol in DCM rendering solubility of CAs, when the final MeOH content decreases from 100% MeOH to 50%,
Fig. 1. Overlay of normalized chromatograms of CAs having different DS (15 mL injection volume, 1.5 g/L of each sample, linear gradient change from 100% DCM to 100% MeOH (a),
100% DCM to 50% MeOH (b), 100% DCM to 35% MeOH (c) and 100% DCM to 13% MeOH (d) in 10 min). The dotted lines represent the eluent composition at the detector.
H.O. Ghareeb, W. Radke / Polymer 54 (2013) 2632e2638 2635
DSi ¼ exp A þ B Ve þ C Ve2 (1)
S DV
wðDSÞ ¼ (2)
Fig. 3. Superimposed chromatograms of CAs having different DS in a multi-step DDS SðS DVÞ
gradient (15 mL injection volume, 1.5 g/L of each sample). The dotted line represents
the eluent composition at the detector.
where S is the ELSD signal which as a first approximation is
assumed to be proportional to concentration. DV is the volume
assignment of the peak maximum to DS obtained by NMR serves difference of two adjacent data points and DDS the difference in DS
therefore only as a first approximation. of two adjacent data points.
Despite some scattering a clear decrease of DS with elution However, the ELSD’s response was reported to be non-linear in
volume is observed. Since the DPw and Ðm of the samples are very concentration and might depend on the nature of the mobile phase
similar (see Table 1) it can be concluded that a separation with and of the solute [49e51]. To investigate the effect of DS on ELSD-
regard to DS is realized within the investigated DS-range for sam- response, identical amounts of the samples were injected without
ples of identical DP. column using pure DCM as eluent. The resulting peak areas are
To elucidate the molar mass contribution to the separation process plotted as a function of average DS in Fig. 5.
under these conditions, samples of similar DS but varying DP are As can be seen, the peak area decreases by approximately 15%
required. However, such samples were not available. In order to get at over the DS-range from DS ¼ 1.5 to DS ¼ 2.9. When this procedure
least some indication on the influence of molar mass on the separa- was repeated for different concentrations, the same dependence of
tion, the elution volumes for two data sets 1 (sample 3, sample 4) and peak area on DS was observed. However, the dependence of peak
2 (sample 13, sample 14, sample 15) having nearly similar DS but area on concentration was linear until an injected mass of approx.
distinct difference in molar mass are inspected more closely. The 0.03 mg, corresponding to the highest injected mass used in the
elution volumes at peak maximum (Vapex) for these samples deter- experiments. Therefore, the dependence of peak area on concen-
mined from Fig. 4 are as follows: Vapex ¼ 36.2 and 35.6 mL for sample 3 tration can be assumed to be linear for the injected mass range used
(Mw ¼ 73,500 g/mol) and sample 4 (Mw ¼ 44,600 g/mol), respectively, in this investigation.
and Vapex ¼ 27.8, 27.0 and 26.8 mL for sample 13 (Mw ¼ 64,400 g/mol), Since the overall change of detector response with DS is rather
sample 14 (Mw ¼ 64,700 g/mol) and sample 15 (Mw ¼ 93,300 g/mol), low, the ELSD signals for the calculation of DS-distribution were
respectively. For samples of identical composition (DS) but varying used without correction.
molar mass, an increase in elution volume with increasing molar From equation (2) the DS-distribution for all of the CA samples
was determined and the results for the selected samples are rep-
resented in Fig. 6.
Table 3
Weight average DS (DSw) calculated from DS-distribution and variance of the DS-
distribution peaks (the samples marked in grey are industrial samples).
Sample name 1
H NMR DS DSw s2
Sample 1 1.53 1.54 0.05
Sample 2 1.59 1.65 0.06
Sample 3 1.66 1.70 0.12
Sample 4 1.72 1.78 0.01
Sample 5 1.81 1.73 0.11
Sample 6 1.87 1.82 0.12
Sample 7 1.92 1.75 0.09
Sample 8 1.95 1.83 0.12
Sample 9 2.09 1.87 0.05
Sample 10 2.16 2.04 0.14
Sample 11 2.19 2.19 0.11
Sample 12 2.27 2.20 0.13
Sample 13 2.42 2.39 0.07
Sample 14 2.45 2.42 0.06
Sample 15 2.45 2.46 0.08
Sample 16 2.60 2.44 0.05
Sample 17 2.92 2.88 0.0007
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