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ISSN 10619348, Journal of Analytical Chemistry, 2015, Vol. 70, No. 1, pp. 32–38. © Pleiades Publishing, Ltd., 2015.

Original Russian Text © N.E. Kuz’mina, S.V. Moiseev, V.I. Krylov, V.A. Yashkir, V.A. Merkulov, 2015, published in Zhurnal Analiticheskoi Khimii, 2015, Vol. 70, No. 1, pp. 30–36.

ARTICLES

Determination of the Average Molecular Weight of Hydroxyethyl


Starches by DiffusionOrdered NMR Spectroscopy
N. E. Kuz’mina, S. V. Moiseev*, V. I. Krylov, V. A. Yashkir, and V. A. Merkulov
Scientific Center for Expertise of Medical Products, Ministry of Health of the Russian Federation,
Petrovskii bul’var 8, Moscow, 127051 Russia
*email: MoiseevSV@expmed.ru
Received October 9, 2013; in final form, February 17, 2014

Abstract—A correlation equation is obtained for the evaluation of the average molecular weight (MW) of
hydroxyethyl starches from the selfdiffusion coefficient D measured using diffusionordered NMR spectros
copy. Correlation equations MW = cD–α are compared for hydroxyethyl starches, dextrans, and pullulans. It
has been demonstrated that different MWs correspond to constant D values of uncharged polysaccharides
with the similar type of nonvalent interactions and different degrees of branching.

Keywords: diffusionordered NMR spectroscopy, average molecular weight of polymers, selfdiffusion coef
ficient, hydroxyethyl starches
DOI: 10.1134/S1061934815010086

INTRODUCTION branched polymer amylopectin consisting of αD


Hydroxyethyl starches (HES) are efficient colloid glucopyranose residues modified with hydroxyethyl
plasma substituents, which are widely used in clinical groups (Scheme). The linear section of the HES mac
practice for the prevention and treatment of hypov romolecules contains α(1 → 4) bonds, branching are
olemia in surgery, traumatology, combustiology, and formed mostly by α(1 → 6) bonds [4, 5]. The side
intensive therapy over the last 50 years [1–3]. The branches of the macromolecule contain from 15 to
molecular structure of HES represents a polydisperse 45 glucose residues.

OH
O O
OH O O
OH
O O
O O
O OH
O O HO
HO O O
HO O O OH
O
HO OH O
OH
OH HO O

OH
Fragment of the molecular structure of HES.

The important characteristics of HES affecting number of hydroxyethyl groups at the sixth carbon
their pharmacokinetic properties are the average atom in the glucopyranose cycle [6–8]. MW deter
molecular weight; molar substitution (MS). which is mines the time of the HES presence in the blood flow.
the ratio of the number of hydroxyethyl groups to the HES macromolecules and fragments of their enzy
total number of glucose molecules; and the ratio matic hydrolysis penetrating through the kidney bar
C2/C6, which characterizes the ratio of the number of rier (45–60 kDa) are rapidly eliminated from the body
hydroxyethyl groups at the second carbon atom to the [7]. Hydroxyethylation slows down the enzymatic

32
DETERMINATION OF THE AVERAGE MOLECULAR WEIGHT 33

hydrolysis of the HES molecules, thus extending the EXPERIMENTAL


time of circulation of the preparation in the blood flow We used HES standard samples produced by the
[8]. A similar effect is made by the high C2/C6 ratio: American Polymer Standard Corporation (United
the higher the C2/C6 ratio, the slower the decomposi 1
tion of HES [6–8]. States) with the average molecular weights Мр 5.5,
8.9, 16.1, 52.8, 80.0,121.1, 153.6, 176.1, 201.0, 219.3,
The determination of the quantitative characteris 330.2, 374.0, 396.9, 468.7, and 1004.0 kDa (HETA
tics of HES (MW, MS, and C2/C6) is a rather complex 6K, HETA 9K, HETA 18K, HETA 60K, HETA 88K,
and laborious problem; in the pharmacopeia analysis HETA 130K, HETA 160K, HETA 175K, HETA
it is solved using a complex of chromatographic meth 230K, HETA 250K, HETA 337K, HETA 380K,
ods: gelpermeation chromatography (GPC) (MW HETA 460K, HETA 600K, and HETA 1300K,
determination), and GC and HPLC (determination respectively). The 1H NMR and 1HDOSY spectra
of MS and C2/C6) [9, 10]. The application of chro were recorded on an Agilent DD2 NMR System 600
matographic methods to the determination of MS and NMRspectrometer with a 5 mm inverse multinuclear
C2/C6 is based on the destruction of the analyzed sensor equipped with a gradient coil at 300 K. The
HES. Therefore, the development of a nondestructive analyzed samples (5 mg) were dissolved in 0.5 mL of
D2O (Cambridge Isotope Laboratories Inc.). The
rapid method that ensures the quick and reliable esti
solutions obtained were diluted to the concentration
mation of all quantitative HES properties using one 1 mg/mL and transferred into Shigemi NMR tubes
sample with the minimal sample preparation is a prob with a glass plunger for the minimization of the effects
lem of current importance. For the development of of convection and the heterogeneity of pulses of the
such a method mostly NMR spectroscopy is used. The magnetic field gradient. For measurements of the self
quantitative determination of MS and sites of HES diffusion coefficient, a DBPPSTE (DOSY Bipolar
substitution was performed based on the data of 1H Pulse Pair Stimulated Echo) sequence was used. Dif
and 13C NMR spectra [4, 11–16]. Methods of tradi fusion attenuation curves were obtained at a sequential
tional NMR spectroscopy are inapplicable to the 15step linear increase of the amplitude of the mag
determination of the MWs of polymers. Diffusion netic field gradient pulse in the range from 1.82 to
ordered NMR spectroscopy (DOSY), which ensures 53 G/cm at fixed values of diffusion time Δ (285 ms),
δpulse gradient pulse duration (2.0 ms), and relaxation
the measurement of the diffusion of various molecular
time d1 (5 s).
objects (molecules, macromolecules, molecular com
plexes, and supramolecular systems) under the impact The results were mathematically processed using
of a magnetic field gradient [17–19], seems to be the the DISCRETE method (Discrete Sum of Exponen
tial Decays). Correlation equation was derived using
most promising method for this purpose. The DOSY the MS Excel 2007 software.
method possesses high potential in the studies of the
molecular weights of polymers [20–23], including
polysaccharides [24–28]. In practice [26–28], the RESULTS AND DISCUSSION
MWs of polysaccharides within the frameworks of the HES is a polydisperse polymer; its MW is an aver
DOSY method were evaluated using the correlation age value and depends on the mode of averaging. In
equation proposed by the authors of [24] for the polymer chemistry, number average (Mn) and weight
dependence of the MW of polysaccharides on the self average (Мw) molecular weights are distinguished. Mn
diffusion coefficient D, which is numerically equal to is obtained by averaging by the number of macromol
the mean square shift of a molecule during the time of ecules in a polymer; Мw is obtained by averaging by the
diffusion td [29]. This equation was obtained on the weight of macromolecules in a polymer. The Mw value
basis of the analysis of linear and lowbranched is sensitive to highmolecular fractions, while Mn is
polysaccharides (dextrans and pullulans). It is known sensitive to lowmolecular weight fractions of polydis
that the topology of a polymer substantially affects its perse polymers; therefore, for the characteristic of the
diffusion, because branched molecules always have average MW of HES we used an averaged parameter
smaller hydrodynamic radii than their linear ana representing molecular weight in peak (Мp), deter
logues [30]; therefore the use of the correlation equa mined by chromatographic methods. For narrowdis
tion for the quantitative evaluation of the MW of HES persed samples Мp = (Mw × Mn)0.5 [31]. The correlation
presented in [24] can lead to incorrect results. relationship between the MW and D values is
described by a power function [24, 27, 32, 33]:
The aim of the present work was to study the effect
of branching on the D value of polysaccharides and the D = cMWα,
evaluation of a correlation relationship between the 1M is the molecular weight of a peak in its maximum in the
p
MW of HES and D taking into account the peculiari determination of the average molecular weight of a polymer by
ties of their steric structures. exclusion gelpermeation chromatography.

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 70 No. 1 2015