Wakopak® Wakosil® GP-N6

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.


  • 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
Flow rate
Injection Volume
: 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


Flow rate
: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

【Pretreatment conditions】
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】
Flow rate
Column switching time
: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

  1. Please use the mobile phase with the pH range of 2.5 to 7.5.
  2. 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.
  3. Pass the sample through a 0.2 μm filter.
  4. 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.
  5. 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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