HPLC Column for Deproteination / Direct Injection of Biological Samples
When analyzing a target compound in a biological sample containing a large amount of protein such as serum by HPLC, deproteinization that requires time and labor is required. Wakopak® Wakosil® GP-N6 is a HPLC column that allows direct injection of protein-containing samples.
In HPLC analysis by Wakopak® Wakosil® GP-N6, macromolecules, such as proteins, pass through the column due to the hydrophilic structure of the modifier, while relatively small molecules, such as drugs, are retained by reverse-phase distribution, etc.
Features
- Capable of direct injection of biological samples
- Excellent durability
- Capable of analysis with high recovery rate and high reproducibility
Comparison with Conventional Pretreatment Procedures
Evaluation of Column Durability
Analysis of blood uric acid
Injection and analysis of 5 μL human serum were repeated. There is almost no change in the peak shape and retention value of uric acid even after 500 analyses, and stable results are delivered.
| First: R.T.-3.96 min., Press.23 kg/cm2 300 analysis: R.T.-3.97 min., Press.27 kg/cm2 500 analysis: R.T.-3.94 min., Press.33 kg/cm2 |
|
| Column Size Eluent Flow rate Detection Sample Injection Volume Peak |
: 4.6 mm x 150 mm : CH3CN/0.2 M Na2HPO4 (pH 6.0)=2/98 (v/v) : 0.5 mL/min. at R.T. : UV 284 nm : Human Serum : 5 μL : 1. Uric Acid |
Example of Direct Analysis
| Column Eluent Flow rate Detection Sample |
：Wakopak® Wakosil GP-N6 (4.6 mm x 150 mm) ：CH3OH/0.1 M Na2HPO4 (pH 6.0)=10/90 (v/v) ：1.0 mL/min. at 40℃ ：UV 254 nm ：1．Acetaminophen 5 μg/mL 2．Sulfamethoxazole 5 μg/mL 3．Ethenzamide 20 μg/mL 4．Salicylamide 25 μg/mL |
Column Switching Method
Wakopak® Wakosil® GP-N6 can be used as a pretreatment column for "column switching analysis", which can be used in pretreatment of biological samples.
Example of analysis of drug contained in serum
| Column Eluent Flow rate |
：Wakopak® Wakosil GP-N6 (4.6 mm x 50 mm) ：CH3CN/0.2 M NaH2PO4, Na2HPO4 (pH 6.0)=2/98 (v/v) ：1.0 mL/min. at 30℃ |
【Analytical conditions】
| Column Eluent Flow rate Detection Column switching time Sample |
：Wakopak® Wakosil-Ⅱ 5C18 RS (4.6 mm x 150 mm) ：CH3CN/0.1 M NaH2PO4, Na2HPO4 (pH 6.0)=30/70 (v/v) ：1.0 mL/min. at 30℃ ：UV 254 nm 0.032AUFS ：4.8-6 min. ：Human Serum 20 μL 1．Phenobarbital 20 μg/mL 2．Carbamazepine 5 μg/mL 3．Phenytoin 40 μg/mL |
Drug recovery rate
| Name of drug | Blood concentration (μg/mL) | Recovery rate (%) | CV (%) |
|---|---|---|---|
| 1. Phenobarbital | 20 | 99.3 | 1.06 |
| 2. Carbamazepine | 5 | 96.5 | 1.12 |
| 3. Phenytoin | 40 | 102.3 | 0.78 |
Precautions on handling
- Please use the mobile phase with the pH range of 2.5 to 7.5.
- Use the organic solvent concentration in the mobile phase at a level that does not denature the proteins (for acetonitrile, we recommend 20% or lower). Do not use solvents that denature the proteins.
- Pass the sample through a 0.2 μm filter.
- To prevent clogging of the column filter, it is recommended that a line filter of approximately 2 μm should be connected between the autosampler and column.
- Please handle the column in the same manner as the reversed-phase columns.
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For research use or further manufacturing use only. Not for use in diagnostic procedures.
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