# Analysis of Chondroitin Sulfate by Size Exclusion Chromatography

August 24, 2022

## Introduction

There are several types (A to E) of chondroitin sulfate (MW 20,000 ~ 50,000) (one of which is a mucopolysaccharide) which are found in animal connective tissue – cartilage and skin. Chondroitin sulfate is widely used in the manufacture of health foods and medicines. In this application note Chondroitin sulfate (sodium salt) was analyzed using a SEC (size exclusion chromatography) column with an exclusion limit of 300,000 molecular weight (Pullulan) and detection using refractive index (RI detector). A Pullulan calibration curve was created from a standard mixture using ChromNAV with the optional GPC Calculation Program and the resulting molecular weights calculated from the Pullulan standard are described below.

## Experimental

### Experiment

Pump: | PU-2080 |

Degasser: | DG-2080-53 |

Column Oven: | CO-2060 |

Autosampler: | AS-2057 |

Detector: | RI-2031 |

### Conditions

Column: | Shodex Asahipak GF-510 HQ (7.5 mmI.D. x 300 mmL, 5 µm) |

Eluent: | 0.04 M Sodium dihydrogen phosphate anhydrous- 0.06 M Disodium hydrogen phosphate anhydrous |

Flow Rate: | 0.6 mL/min |

Column Temp.: | 30 ºC |

Injection Volume: | 50 µL |

Calibration standards for SEC: | Shodex STANDARD P-82 (Pullulan) 0.05 % each eluent |

### Structure

## Keywords

HPLC, size exclusion, chondroitin sulfate, SEC, RI detector, GPC Calculation, molecular weight

## Results

Fig. 1 Chromatograms of Pullulan (PL) standard mixture for molecular weight calibration and chondroitin sulfate sodium salt.

Fig. 2 Molecular weight calibration curve created with a mixed Pullulan standard.

Fig. 3 Chromatogram of chondroitin sulfate sodium salt and the molecular weight calibration curve.

Fig. 4 and Table 1 show the molecular weight distribution calculation curve and the molecular weight as calculated using a Pullulan standard.

### Hint

**Molecular weight calibration curve**

Shows the relationship between retention volume (elution time) and molecular weight, which is created to estimate the molecular weight of the sample from its retention volume (elution time).

**Molecular weight distribution curve**

The integral molecular weight distribution curve shows the relationship between molecular weight (logarithmic value) and the percentage of the total of a specific molecular weight.

The differential molecular weight distribution curve is used to plot the molecular weight (logarithmic value) on the x-axis against the weight fraction differentiated from a logarithmic value of molecular weight (dw/d(logM)) on the y-axis. When normalized, the use of this curve makes it possible to compare a chromatogram and a molecular weight distribution derived using different columns and measurement conditions.

**Molecular weight averages and polydispersity**

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 weights. These averages are defined by the following expressions. The distribution of these molecular weight averages bears in general a relationship such as Mn ? Mv ? Mw ? Mz. In the case where Mn = Mv = Mw = Mz, there is no molecular weight distribution (mono-dispersed).

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## Analysis of Chondroitin Sulfate by Size Exclusion Chromatography

### Introduction

There are several types (A to E) of chondroitin sulfate (MW 20,000 ~ 50,000) (one of which is a mucopolysaccharide) which are found in animal connective tissue – cartilage and skin. Chondroitin sulfate is widely used in the manufacture of health foods and medicines. In this application note Chondroitin sulfate (sodium salt) was analyzed using a SEC (size exclusion chromatography) column with an exclusion limit of 300,000 molecular weight (Pullulan) and detection using refractive index (RI detector). A Pullulan calibration curve was created from a standard mixture using ChromNAV with the optional GPC Calculation Program and the resulting molecular weights calculated from the Pullulan standard are described below.

### Experimental

### Experiment

Pump: | PU-2080 |

Degasser: | DG-2080-53 |

Column Oven: | CO-2060 |

Autosampler: | AS-2057 |

Detector: | RI-2031 |

### Conditions

Column: | Shodex Asahipak GF-510 HQ (7.5 mmI.D. x 300 mmL, 5 µm) |

Eluent: | 0.04 M Sodium dihydrogen phosphate anhydrous- 0.06 M Disodium hydrogen phosphate anhydrous |

Flow Rate: | 0.6 mL/min |

Column Temp.: | 30 ºC |

Injection Volume: | 50 µL |

Calibration standards for SEC: | Shodex STANDARD P-82 (Pullulan) 0.05 % each eluent |

### Structure

### Keywords

HPLC, size exclusion, chondroitin sulfate, SEC, RI detector, GPC Calculation, molecular weight

### Results

Fig. 1 Chromatograms of Pullulan (PL) standard mixture for molecular weight calibration and chondroitin sulfate sodium salt.

Fig. 2 Molecular weight calibration curve created with a mixed Pullulan standard.

Fig. 3 Chromatogram of chondroitin sulfate sodium salt and the molecular weight calibration curve.

Fig. 4 and Table 1 show the molecular weight distribution calculation curve and the molecular weight as calculated using a Pullulan standard.

### Hint

**Molecular weight calibration curve**

Shows the relationship between retention volume (elution time) and molecular weight, which is created to estimate the molecular weight of the sample from its retention volume (elution time).

**Molecular weight distribution curve**

The integral molecular weight distribution curve shows the relationship between molecular weight (logarithmic value) and the percentage of the total of a specific molecular weight.

The differential molecular weight distribution curve is used to plot the molecular weight (logarithmic value) on the x-axis against the weight fraction differentiated from a logarithmic value of molecular weight (dw/d(logM)) on the y-axis. When normalized, the use of this curve makes it possible to compare a chromatogram and a molecular weight distribution derived using different columns and measurement conditions.

**Molecular weight averages and polydispersity**

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 weights. These averages are defined by the following expressions. The distribution of these molecular weight averages bears in general a relationship such as Mn ? Mv ? Mw ? Mz. In the case where Mn = Mv = Mw = Mz, there is no molecular weight distribution (mono-dispersed).