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Separation of C1 Compounds (DE-413)

RSpak ​DE-413, a polymer-based reversed phase chromatography column, can be used with aqueous eluents with no organic solvent. In this application, simultaneous analysis of C1 compounds including formaldehyde, methanol, and formic acid was performed using an aqueous solution of phosphoric acid as an eluent.

Sample: 20 μL
2.5 mg/mL each (in H2O)

1.Formaldehyde

2.Methanol

3.Formic acid

C1_ompounds_chromatogram
 	Column：Shodex RSpak DE-413 (4.6 mm I.D. x 150 mm) x 2
 	Eluent：10 mM H3PO4 aq.
 	Flow rate：1.0 mL/min
 	Detector：RI, UV (190 nm)
 	Column temp.：50 ℃

Separation of Furfural and Guaiacol (DE-413)

Wine contains furfural and guaiacol. Furfural has butterscotch and caramel notes, and guaiacol has a charcoal, smoky note, both contribute to wine flavoring. In this application, furfural and guaiacol were analyzed using RSpak DE-413, a polymer-based reversed phase chromatography column.

Sample: 20 µL

1.20 μg/mL Furfural (2-Furaldehyde)

2.10 μg/mL Guaiacol (2-Methoxyphenol)

Furfural_Guaiacol_chromatogram
 	Column：Shodex RSpak DE-413 (4.6 mm I.D. x 150 mm)
 	Eluent：2 mM HClO4 aq./CH3CN=50/50
 	Flow rate：1.0 mL/min
 	Detector：UV (210 nm)
 	Column temp.：40 ℃

Analysis of Chelating Agents (ODP-50 4D)

Three chelating agents were analyzed using Asahipak ODP-50 4D, a polymer-based reversed phase chromatography column. The chelating agents are pretreated to convert into chelating compounds (iron chelates) before analysis.

Sample pretreatment

  • Dissolve chelating agents in a mixture of 10 mM sodium carbonate, 10 mM sodium bicarbonate, and 10 mM tetra-n-butylammonium hydroxide solution (pH 11) for an efficient complex salt formation.
  • Add an excess amount of 0.01 M ferric chloride solution*, approximately about 2 mL of 0.01 M ferric chloride solution to 20 mL of 100 ppm solution.
    *Dissolve 0.135 g of ferric chloride in 5 mL of 1 M hydrochloric acid aqueous solution, then make up to 50 mL with water.
（Note）
Once 0.01 M ferric chloride solution is added, excess iron precipitates as ferric hydroxide. It is recommended to analyze samples immediately after adding the 0.01-M ferric chloride solution.

Sample: 100 mg/L each, 10 μL

1.2-Hydroxyethyl)ethylenediamine-N,N’,N’-triacetic acid (HEDTA)

2.Ethylenediaminetetraacetic acid disodium salt (EDTA·2NA)

3.Diethylenetriaminepentaacetic acid dianhydride (DTPA)

Chelating_ agents_chromatogram
Column：Shodex Asahipak ODP-50 4D (4.6 mm I.D. x 150 mm)Eluent：10 mM Na2CO3 + 10 mM NaHCO3 + 10 mM Tetrabutylammonium hydroxide (pH7.0 adjusted with 1 M HCl)
Flow rate：0.8 mL/min
Detector：UV (254 nm)
Column temp.：40 ℃

Analysis of Residual ε-Caprolactam in Nylon 6 (ODP-50 4D)

ε-caprolactam, a residual monomer in nylon 6, was analyzed using Asahipak ODP-50 4D, a reversed-phase chromatography column. Analysis of ε-caprolactam can be performed without an influence from extract contaminants.

Sample pretreatment

  • Add 10 mL of pure water to 0.5 g of nylon 6 pellets. Seal and heat at 100 ℃ for 2 hours.
  • Once the mixture is cooled to room temperature, filter through a 0.5μm filter. Use the filtrate as a test sample.

Sample: 10 μL

1.ε-Caprolactam

ε-Caprolactam_chromatogram
 	Column：Shodex Asahipak ODP-50 4D (4.6 mm I.D x 150 mm)
 	Eluent：H2O/CH3CN=80/20
 	Flow rate：0.7 mL/min
 	Detector：UV (210 nm)
 	Column temp.：40 ℃

Analysis of Macrolide Antibiotics and Derivatives (ODP-50 4D)

Asahipak ODP-50 4D, a reversed phase chromatography column, was used for simultaneous analysis of erythromycin, a macrolide antibiotics, and its derivative azithromycin. Since macrolide antibiotics are basic substances, their dissociation is suppressed under alkaline conditions, and this makes them easily retained in reversed phase analysis. ODP-50 4D has an excellent  durability towards alkaline conditions, and thus enables this analysis.

Sample: 0.1 % each, 10 μL

1.Erythromycin

2.Azithromycin

Macrolide_antibiotics_chromatogram
Column：Shodex Asahipak ODP-50 4D (4.6 mm I.D x 150 mm)
Eluent：40 mM Potassium phosphate buffer (pH11.0)/CH3CN=40/60
Flow rate：0.5 mL/min
Detector: UV (223nm)
Column temp.：40 ℃

 

 

The reversed phase Asahipak ODP-40 4E column was used here to analyze simultaneously the macrolide antibiotic Erythromycin and its derivative Azithromycin. The polymer-based ODP-40 4E column is very alkaline-resistant and can therefore be used under alkaline conditions. *Sales of the product used in this application is discontinued, however if you are still interested in this application, please contact us. s1431 Sample : 10 μL 0.1 % each 1. Erythromycin 2. Azithromycin

Column       : Shodex Asahipak ODP-40 4E (4.6 mm I.D. x 250 mm)
Eluent       : 40 mM Potassium phosphate buffer (pH11.0)/CH3CN=40/60
Flow rate    : 0.5 mL/min
Detector     : UV (223 nm)
Column temp. : 40 °C

Analysis of 3,4-Dimethylpyrazole Phosphate According to Japanese Testing Methods for Fertilizers (C18M 4D)

3,4-Dimethylpyrazole phosphate (DMPP) is a nitration inhibitor added to solid fertilizers. The Japanese regulation for “Testing Methods for Fertilizers (2022*)” mentions to use an octadecylsilylated silica gel column for DMPP analysis. A calibration curve for DMPP obtained using Silica C18M 4D showed a high linearity with a coefficient of determination (R2) 0.9999 in the concentration range 0.5 to 50 μg/mL.
Urea fertilizer, one of the target fertilizers, was analyzed. The extract was prepared in accordance with the Testing Methods for Fertilizers. DMPP was not detected in the extract of the fertilizer analyzed.  As a next step, addition recovery test was performed using the same sample (spiked DMPP concentration 0.185 mg/g). A good result was obtained with an average recovery rate of 102 % (n=3).

*The version at the time of the application acquisition.

Sample preparation

  1. (1)Measure 1.00 g of urea fertilizer and put it in a 200-mL erlenmeyer flask. (For the spike recovery test DMPP was added to the sample at this point.)
  2. (2)Add 100 mL of water to (1) and stir with a magnetic stirrer for 10 minutes.
  3. (3)After allowing it to stand, transfer a portion of the supernatant to a 1.5-mL glass-stoppered centrifugal sedimentation tube.
  4. (4)Centrifuge (8000 ~ 10000 x g for about 5 minutes) and use the supernatant as an injection sample.

Sample: 10 µL
Standard solution (5 µg/mL)
Sample solution spiked with DMPP (0.185 mg/g)
Sample solution 1.DMPP (3,4-dimethylpyrazole phosphate)

DMPP_chromatogram

DMPP_carlibration _curve
 	Column：Shodex Silica C18M 4D (4.6 mm I.D x 150 mm)
 	Eluent：10 mM NaH2PO4 aq./CH3CN=1000/175
 	Flow rate：0.7 mL/min
 	Detector：UV (224 nm)
 	Column temp.：40 ℃

Separation of Ceftazidime and Arginine (KW402.5-4F)

Ceftazidime is a cephem antibiotic used to treat bacterial infections. Some formulations of ceftazidime contain arginine. KW402.5-4F, an aqueous SEC (GFC) column, was used to separate ceftazidime and arginine, and a good separation was obtained.

Sample: 0.2 mg/mL each, 10 µL

1.Ceftazidime

2.L-Arginine

Ceftazidime_chromatogram
 	Column：Shodex KW402.5-4F (4.6 mm I.D. x 300 mm)
 	Eluent：1.15 g/L NH4H2PO4 (pH 2.0 adjusted with H3PO4)/CH3CN=25/75
 	Flow rate：0.5 mL/min
 	Detector：UV (206 nm)
 	Column temp.：30 ℃

Analysis of Carboplatin (NH2P-50 4E)

Carboplatin, a type of anticancer drug, is called a platinum agent because it contains platinum in its structure. A silica-based amino column is used in Assay of Carboplatin listed in the United States Pharmacopeia and the National Formulary (USPNF 2022 Issue 1*). With reference to this USP monograph, carboplatin was analyzed using Asahiak NH2P-50 4E, a polymer-based amino column. We confirmed that NH2P-50 4E was also suitable for the analysis.

*The version at the time of the application acquisition.

Sample: 10 µL

1.Carboplatin 1 mg/mL

Carboplatin_chromatogram
 	Column：Shodex Asahipak NH2P-50 4E (4.6 mm I.D. x 250 mm)
 	Eluent：CH3CN/H2O=83/17
 	Flow rate：1.5 mL/min
 	Detector：UV (230 nm)
 	Column temp.：25 ℃

Analysis of High Molecular Weight Protein According to USP-NF GENERAL CHAPTERS Physicochemical Analytical Procedures for Insulins (KW-802.5)

USP-NF GENERAL CHAPTERS lists <121.1> Physicochemical Analytical Procedures for Insulins. The target analytes included in this method are insulin analogues, animal-derived insulin, and human insulin. The analysis of high molecular weight proteins should be carried out with a column filled with L20 packing material and meets following requirements.

 

System suitability requirements

Retention times:
Polymeric insulin complexes: 13 – 17 min
Covalent insulin dimer: about 17.5 min
Insulin monomer: 18 – 22 min
Peak-to-valley ratio*: ≥ 2.0
*The ratio of the height of the covalent insulin dimer peak to the height of the valley between the covalent insulin dimer peak and the insulin monomer peak.

Below lists the monographs using this method (at 2023 June).

Insulin Insulin Injection Insulin Human Insulin Human Injection
Insulin Aspart Insulin Aspart Injection Insulin Glargine Insulin Glargine Injection
Insulin Lispro Insulin Lispro Injection Insulin Zinc Suspension Extended Insulin Zinc Suspension
Prompt Insulin Zinc Suspension Isophane Insulin Suspension Isophane Insulin Human Suspension Human Insulin Isophane Suspension
and Human Insulin Injection

The PROTEIN KW-802.5 confirmed the requirements were met for the analysis of insulin beef.

Sample: 100 µL
4.0 mg/mL of Insulin (beef) containing dimer (in 0.01 N HCl aq.)

  1. 1.High molecular weight proteins
  2. 2.Insulin dimer
  3. 3.Insulin monomer
s1664
Column：Shodex PROTEIN KW-802.5 (8.0 mm I.D. x 300 mm)
Eluent：0.1 wt% L-Arginine aq./CH3CN/CH3COOH=13/4/3
Flow rate：0.5 mL/min
Detector：UV (276 nm)
Column temp.：25 ℃

Analysis of Proteoglycan in a Supplement (SB-806M HQ)

Proteoglycan, a type of glycoprotein, is found in cartilages of humans and animals. They are glycosaminoglycans bound to proteins. Proteoglycan promotes skin cell growth and the production of hyaluronic acid and collagen. Proteoglycan itself also has a high-water retention capability. Using those advantages, proteoglycans are used in functional foods and cosmetics, expecting the effects such as anti-aging of the skin, alleviation of arthritis symptoms, and inflammation suppression.
In this application, proteoglycan in a commercial supplement was analyzed using OHpak SB-806M HQ, an aqueous SEC column. A good linear calibration was achieved. Analysis of a commercial supplement showed a close to a 100 % recovery rate, compared to the manufacturer’s information provided.

Sample preparation

  1. Grind a supplement in a mortar to make it into powders.
  2. Measure 270 mg of the powder and add 15 mL of 4-M guanidine hydrochloride solution.
  3. Sonicate for 15 minutes and then centrifuge at 1800 x g for 10 minutes.
  4. Collect the supernatant. Add another 15 mL of 4-M guanidine hydrochloride to the residue and repeat step (3). Repeat this process one more time (i.e., a total of 3 times adding the 4-M guanidine hydrochloride).
  5. Ultrafiltrate (MW: 50,000-100,000) all collected supernatant from step (4) at 2400 x g for 30 minutes.
  6. Add 5 mL of water to the “proteoglycan fraction” which did not pass through the ultrafiltration membrane. Centrifuge at 1800 x g for 10 minutes. Repeat this step five times to wash the proteoglycan fraction.
  7. Add eluent to the proteoglycan fraction to make up the volume of 20 mL.
  8. Filtrate with 0.45μm membrane and use it as an injection sample.

 

Sample: 50 µL
Proteoglycan standard solution (0.1 mg/mL)
Pretreated supplement extract solution

  1. Proteoglycan
Proteoglycan_chromatogram
Proteoglycan_calibration curve
 Columns : Shodex OHpak SB-806M HQ (8.0 mm I.D. x 300 mm) + SB-G 6B (6.0 mm I.D. x 100 mm) 
 Eluent : 25 mM KH2PO4 + 25 mM Na2HPO4 aq.
 Flow rate : 0.5 mL/min
 Detector : RI
 Column temp. :40 ℃