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Analysis of Ellagic Acid in a Supplement (C18U 2D)

Ellagic acid, a type of natural polyphenol contained in pomegranate and raspberries, is a functional food component. It has various effects such as reduction of body fat, blood triglyceride, and visceral fat, or works as an antioxidant, antiviral, and skin whitening reagent. In this application, ellagic acid in a commercial supplement was analyzed using a UHPLC column, C18U 2D. A good linear calibration was obtained for the concentration range of 0.1 to 5 mg/L.

(Sample Preparation)
1) Grind a dietary supplement tablet in a mortar to make it into powders.
2) Precisely measure 0.2 g of the powder and add 15 mL of dimethyl sulfoxide.
3) Sonicate for 10 minutes and then centrifuge at 2500 rpm for 5 minutes.
4) Collect the supernatant. Add another 15 mL dimethyl sulfoxide and repeat (3).
5) Fill up the collected supernatant with dimethyl sulfoxide to make up the volume to 50 mL.
6) Take 1 mL out from (5) and dilute with dimethyl sulfoxide to make up the volume to 10 mL. Use it as an injection sample.

Sample: 2 μL each
Standard 1 mg/L (in DMSO)
1. Ellagic acid

Analysis of Ellagic Acid in a Supplement C18U 2D

Column       : Shodex C18U 2D (2.0 mm I.D. x 150 mm)
Eluent       : 0.1 % H3PO4 aq./CH3OH=60/40
Flow rate    : 0.2 mL/min
Detector     : UV (254 nm)
Column temp. : 40 °C

 

Absolute Molecular Weight Determination of PMMA (LB-802.5)

The absolute molecular weight of PMMA was determined by OHpak LB-802.5, a column for aqueous SEC. LB-800 series are compatible with multi-angle light scattering (MALS) detectors by minimizing the baseline noise level.

Sample: 50 μL
0.1 % PMMA

Absolute Molecular Weight Determination of PMMA LB 802.5

Columns      : Shodex OHpak LB-802.5 (8.0 mm I.D. x 300 mm)
Eluent       : CH3CN
Flow rate    : 1.0 mL/min
Detector     : RI + MALS
Column temp. : 30 °C

Effects of Acetonitrile in Eluent (2) (KC-811)

When analyzing organic acids using RSpak KC-811, the elution time of organic acids with strong hydrophobicity can be shortened by adding 10 % acetonitrile to the eluent.

s0179 sharpSample:

1.Succinic acid
2.Formic acid
3.Lactic acid
4.Acetic acid
5.Propionic acid
6.Isobutyric acid
7.n-Butyric acid
8.Isovaleric acid
9.n-Valeric acid

Column:Shodex RSpak KC-G 8B (8.0 mm I.D. x 50 mm) + KC-811 (8.0 mm I.D. x 300 mm) x 2
Eluent:2 mM HClO4 aq. or 2 mM HClO4 aq./CH3CN=90/10
Flow rate:(Eluent); 1.0 mL/min, (Reagent); 1.1 mL/min 
Detector:VIS (430 nm) *Post column method
Column temp.:47 ℃ 

Calibration Curves for HK-400 Series (HFIP Eluent : PMMA)

Calibration curves for the GPC HK-400 series, and organic SEC (GPC) columns for ultra-rapid analysis, using PMMA are shown.

Sample : Poly(methyl methacrylate)

Calibration Curves for HK 400 Series

Columns      : Shodex GPC HK-400 series (4.6 mm I.D. x 150 mm each)
Eluent       : 5 mM CF3COONa in HFIP
Flow rate    : 0.3 mL/min
Detctor      :  RI (small cell volume)
Column temp. : 40 °C

 

Analysis of Polyhydroxybutyric Acid (LF-804)

Polyhydroxybutyric acid is a 3-hydroxybutyric acid (ketone body) polyester. It has been focused because of its function as a biodegradable plastic. Recently, its applications in foods and pet foods are also expected. In this application, polyhydroxybutyric acid was analyzed using an organic SEC (GPC) column, GPC LF-804. Chloroform was used as an eluent.

 

Sample: 100 µL
Polyhydroxybutyrate 0.2 %

Analysis of Polyhydroxybutyric Acid

 

Column:Shodex GPC LF-804 (8.0 mm I.D. x 300 mm) 
Eluent:CHCl3
Flow rate:1.0 mL/min
Detector:RI
Column temp.:40 ℃

Effects of Column Unit on Separation (HK-406: PS)

Polystyrene (Mp 580) was analyzed using GPC HK-406, an organic SEC (GPC) column for ultra-rapid analysis. Different numbers of HK-401 columns (1, 2, and 3 units) were used alone/together in series to compare their separation efficiencies. The use of multiple columns improves the separation of oligomers, although it also increases the analysis time. Regardless, the analysis time using three HK-406 columns takes less than 12 minutes.

Sample: 0.5 %, 5 µL
1. Polystyrene (Mp: 609,000)
2. Polystyrene (Mp: 53,000)
3. Polystyrene (Mp: 3,180)

 

Effects of Column Unit on Separation

Columns      : Shodex GPC HK-406 (4.6 mm I.D. x 150 mm) x n
Eluent       : THF
Flow rate    : 0.6 mL/min
Detector     : RI (small cell volume)
Column temp. : 30 °C

Epoxy Resin (7) ( Effects of Flow Rate for HK-402)

GPC HK-400 series, organic SEC (GPC) columns for ultra-rapid analysis have low column pressure even at high flow rates and can be used in conventional HPLC devices. The separation of epoxy resin (jER1001) using HK-402 was compared at flow rates of 0.3 mL/min and 1.0 mL/min. At a flow rate of 1.0 mL/min, equivalent separation can be achieved with an analysis time of 1/6 or less of a conventional SEC column, but if separation is important, it is recommended to analyze at 0.3 mL/min. (Even at 0.3 mL/min, the analysis time is less than half that of a conventional SEC column.)
Note: Epoxy Resin (6) (Comparison between HK-402 and KF-802.5)

Sample: 10 µL
jER1001 (Epoxy resin) 0.2 %(w/v)

Epoxy Resin 7

 

Column       : Shodex GPC HK-402 (4.6 mm I.D. x 150 mm)
Eluent       : THF
Flow rate    : 0.3 mL/min, 1.0 mL/min
Detector     : UV (254 nm) (small cell volume)
Column temp. : 40 ℃

Analysis of Coenzyme Q10 in a Supplement (C18M 4D)

Coenzyme Q10, also known as ubiquinone Q10 (or ubiquinone), is a fat-soluble substance found in foods such as meat and seafood. It is expected to have antioxidant and anti-aging effects and is also known as a vitamin-like substance. In this application, we analyzed a commercially available supplement (soft capsule) using a silica-based reversed-phase chromatography column, Silica C18M 4D. C18M 4D was feasible analyzing coenzyme Q10 without having interference from other components. In addition, obtained calibration curve prepared using standard samples showed a highly linear calibration curve with a coefficient of determination (R2) 0.9991, in the concentration range of 10 to 1000 mg/L.

Sample pretreatment

  1. Remove the powder contents of the supplement (soft capsule).
  2. Add 4 mL of eluent to 5 mg of the sample. Apply sonication for 10 minutes, and then heat at 40℃ for 2 minutes to dissolve the sample completely.
  3. Let it cool to room temperature and make up to 5 mL with eluent.
  4. Filter with 0.45 µm filter and use it as a sample.

Sample: 5 μL
(Sample solution)
(Standard solution) 1000 mg/L in eluent

  1. Coenzyme Q10
chromatogram of Coenzyme Q10
calibration of Coenzyme Q10

 

Column:Shodex Silica C18M 4D (4.6 mm I.D. x 150 mm)
Eluent:CH3OH/C2H5OH=40/60
Flow rate:1.0 mL/min
Detector:UV (275 nm)
Column temp.:35 ℃

Analysis of Resveratrol in Wines (C18M 4D)

Resveratrol is a type of polyphenol found primarily in grape and peanut skins. Resveratrol has antioxidant effects and is expected to have effects such as anti-aging, prevention of diabetes, and improvement of blood pressure. In this application, we analyzed resveratrol in commercially available white and red wine (after sample pretreatment steps) using a silica-based reversed-phase chromatography column, The obtained calibration curve prepared using standard samples showed a highly linear calibration curve with a coefficient of determination (R2) 1.0000, in the concentration range of 0.01 to 10 mg/L.
A resveratrol peak was detected in red wine, but only a small peak was observed in white wine. It can be considered that in general, red wine contains larger amount of resveratrol since the grape juice is squeezed out together with the skins and seeds, meanwhile white wine contains less resveratrol because the grape juice is squeezed out after removing the skins and seeds from the grapes.

Sample pretreatment

  1. (Measure 50 mL of commercial wine and add 40 mL of water. Adjust the pH to 7.0 with 1 M NaOH aqueous solution.
  2. Make up to 100 mL with Water.
  3. Condition a polymer-based reversed-phase octadecyl functional group solid phase cartridge by passing 10 mL of methanol, and 50 mL of 30 mM sodium phosphate buffer (pH7.0).
  4. Pour the wine sample from the step (2). (10 mL of red wine or 50 mL of white wine)
  5. Blow nitrogen gas onto the solid phase cartridge to dry.
  6. Pour 5 mL of ethyl acetate into the solid phase cartridge and collect the entire elute.
  7. Blow nitrogen gas onto the obtained solution to dry.
  8. Dissolve the obtained powder with a small volume of eluent. Make up with the eluent, up to 5 mL for red wine or 1 mL for white wine.
  9. Filter with 0.45 µm filter and use it as a sample.

Sample: 5 μL
(Pretreated sample solution)
Red wine, White wine
(Standard solution) 1 mg/mL Resveratrol in eluent

  1. Resveratrol
chromatogram of Resveratrol
calibration of Resveratrol
Column:Shodex Silica C18M 4D (4.6 mm I.D. x 150 mm)
Eluent:(A); 10 mM H3PO4 aq./CH3CN=80/20
    (B); 10 mM H3PO4 aq./CH3CN=50/50
    Linear gradient; (B %) 0 % (0 to 14 min), 0 % to 100 % (14 to 14.01 min), 100 % (14.01 to 20 min), 0 % (20.01 to 40 min) 
Flow rate:1.0 mL/min
Detector:UV (303 nm)
Column temp.:40 ℃

Analysis of PMMA in reference to USP-NF GENERAL CHAPTERS “Molecular Weight Determination for Copolymers Containing Alkyl Methacrylate or Alkyl Acrylate” (LF-804)

The GENERAL CHAPTERS of the United States Pharmacopeia and the National Formulary (USP-NF 2023 ISSUE 3*) includes “<314> Molecular Weight Determination for Copolymers Containing Alkyl Methacrylate or Alkyl Acrylate”. The method for “apparent weight-average molecular weight and polydispersity” measurement is carried out with a column filled with L128 packing material and meets following requirement. The use of GPC LF-804 column was used in this application, using the above-mentioned method as a reference and confirmed the requirements were met.

System suitability requirement

  1. Correlation coefficient of the calibration curve (created using 7 or more polymethyl methacrylate (PMMA) standard samples with different molecular weights in the peak top molecular weight range of approximately 600 to 2,000,000 Da (g/mol)): ≥ 0.99
  2. Relative standard deviation of the weight average volume (determined from the calibration curve in six replicate analyzes using a system suitability solution (approximately 190,000 Da PMMA standard solution)): ≤ 10%.

*The version at the time of the application acquisition.

Sample: 20 μL
(Standard solution) 1.0 mg/mL each

  1. PMMA (Mp 965,000)
  2. PMMA (Mp 505,000)
  3. PMMA (Mp 224,000)
  4. PMMA (Mp 66,700)
  5. PMMA (Mp 20,100)
  6. PMMA (Mp 6,140)
  7. PMMA (Mp 2,870)
chromatogram of PMMA
calibration of PMMA

(System suitability solution) 1.0 mg/mL PMMA (Mp 211,000)

chromatogram of PMMA

Currently, LF-804 is not included in L128.

 

Column:Shodex GPC LF-804 (8.0 mm I.D. x 300 mm) x 2
Eluent:THF
Flow rate:1.0 mL/min
Detector:RI
Column temp.:30 ℃