硫酸软骨素的Size Exclusion Chromatography

硫酸软骨素（分子量20000～50000）（其中一种是粘多糖）存在于动物结缔组织软骨和皮肤中，有几种类型（A至E）。硫酸软骨素广泛应用于保健食品和药品的生产。在本申请中，硫酸软骨素（钠盐）使用排除极限为300000分子量的SEC（Size Exclusion Chromatography）柱（Pullulan）进行分析，并使用折射率（RI检测器）进行检测。使用ChromNAV和可选的GPC计算程序从标准混合物中创建Pullulan校准曲线，并且由普鲁兰标准计算得到的所得分子量如本应用所述。Application Note 2/4 Analysis of Chondroitin Sulfate Sodium Salt by SEC C Introduction There are several types (A to E) of chondroi ti n sulfate (MW 20，000~50，000) (one of which is a mucopolysaccharide)which are found in ani m al connective tissue -cartilage and ski n . Chondroitin sulfate is widely used in t he manufacture of health foods and medicines. In this application note Chondroitin sulfate (sodium salt ) was ana l yzed using a SEC (Size Exc l usion Chromatography) column with an excl u sion limit of 300，000molecular weight (Pullulan) and detection using refract i ve index (RI detector). A Pul l ulan calibration curve was created from a standard mixture using ChromNAV with the optional GPC Calcul a tion Program and t he resulting molecular weights calculated from the Pullulan stand a rd are described below. JASCO Re fr act iv e Index Detector at www.jascoi n c.co m 28600 Mary's Court， Easton， MD 21601 USA Analysis of Chondroitin Sulfate Sodium Salt by SEC Application Note Exper i mental Equipment Conditions Pump： PU-2080 Degasser： DG-2080-53 Column Oven： CO-2060 Autosampler： AS-2057 Detector： RI-2031 Column： Shodex Asahipak GF-510HQ (7.5 mml.D.x300 mmL， 5) um Flowrate： Column Temp.： Structure n PL-STD1 PL-STD2 Grade Mp Grade Mp P-100 P-20 P-5 107，000 21，100 5，900 P-200P-50 P-10 200，000 47，100 9，600 Chondroi ti n Sulfate C Sample： 0.1% Chondroit i n sul f ate C sodium salt in eluent Results Fig. 1 shows the chromatograms of Pullulan (PL)， standard mixture for molecular weight calibrat i on and chondroi ti n sulfa t e sod iu m s a lt. Fi g. 1. Chromatograms of Pullulan (PL )， Standard Mixtur e for Molecular Weight Calibration and Chondroi t in Sulfat e Sodium Sali (Each value o n t h e chromat ogr ams is Mp (Peak top m olecular weig ht).) Fig. 2 shows molecular weight calibration curve created with Pullulan as a standard mixture. Fig. 3 shows a chromatogram of chondroitin sulfate sodium salt and molecular weight calibration curve. Fig. 4 and Table 1 shows molecular weight distribution calculation curve and the result of molecular weig h t calculated with Pullulan respectively. Fig . 2. Molecular Weight Calibrat i on Curve Created with PL Standard Mixture Fig. 3. Chromatogram of Ch o ndroit i n Sulfate Sodium Salt a n d Molecular Weight Cali b rat i on Curve (T he f igure i n blue represents Mp calculated with Pullulan .) Fig . 4. Mol e cular We i ght Distribution Curve of Chondroitin Sulfate Sodium Salt T able 1. Pullulan Conve r ted Molecular Wei gh t of Chondroit i n Sulfate Sodium Salt Mp Mn Mw Mz Mv Mw/Mn Mz/Mw 46812 36668 57914 85947 57914 1.58 1.48 Shows the relationship between retention volume (elution time) and molecular weight ， which is created to estimate the mo l ecular we i ght of t he sample from its retention volume (elution time). The integral mo l ecular weight distribution curve shows the relat i onship between molecul a r weig h t (logarithmic value) and th e percentage of the total of a speci f ic molecular weight . The differential molecular weight distribut i on curve is used to plot t he molecular weight (logarithmic value) on the x -axis against the weight f rac t ion differentiated f rom a logarithmic value of molecular weight (dw/d(logM))on t he y -axis. When normal i zed， the use of t his curve makes it possible to compare a chromatogram and a molecular weight distribution de r ived us i ng different columns and measurement conditions. The molecular weight averages of polymer materials obtained by size exclusion chromatography (SEC)includes the number-average (Mn)， weight average (Mw)， z-average (Mz)， and viscosity-average (Mv) molecular weig h ts . These averages are defined by the followi n g expressions. The distribution of these molecular weight averages bears in general a relationship such as Mn ≤ Mv ≤ Mw < Mz. I n case of Mn =Mv =Mw = Mz， there is no m olecular weight distribu t ion (mono-dispersed). -1/a *N. represents t he number of molecules for i component of molecular weight M， and a represents an index of Mark-Houwink-Sakurada equation.