(Copy) Wakopak^® Wakosil^®-II Prep Series (C18, SIL) (for Preparative Chromatography)

Wakopak^® Wakosil^®-II Prep Series Features

• High purity silica gel with low metal content is used.
• Unique features of the series (AR, HG, RS) are available.
• Packing agents with the same separation characteristics are available from analysis to preparative size (packed with the same packing agent).

*1 Wakosil^®-II HG Prep: The highest number of theoretical plates among our column products.
*2 Wakosil^®-II RS Prep: High retention capacity due to its large specific surface area. Because of its thorough end capping treatment, it has high separation and retention capacity even with aqueous eluents, making it suitable for the separation of highly polar compounds.
*3 Wakosil^®-II AR Prep: This product has excellent stereoselectivity due to its polymeric type chemical modification. Also, the mobile phase can be used over a wide pH range due to its extremely high durability.
*4 Prep series columns are available in 4.6, 10, 20, and 50 mm I.D. columns. For sizes not listed in the product list, please contact us.

End Capping

The reaction between the silanol groups of silica gel and silylating agent is affected by steric hindrance and other factors, so some silanol groups may remain. These residual silanol groups can cause peak tailing of basic compounds and affect separation. Further silylation to remove the residual silanol groups is called end capping. Silane compounds such as trimethylchlorosilane (TMS) are mainly used for end capping.

[Example of end capping on an ODS column]

Monomeric and Polymeric

Monomeric refers to a structure in which the silylating agent and the silanol group on the silica gel are bonded one-to-one. Polymeric refers to a structure in which multifunctional silylating agents polymerize with each other or one silylating agent bonds to silica gel at multiple silanol groups during the ODS modification of silica gel, forming a dense octadecyl layer.

[Monomeric]

[Polymeric]

Consideration of optimum separation conditions in a preparative column requires a large number of samples, and the preparative columns are expensive. Therefore, the usual approach is to set the conditions in advance on an analytical size column and transfer them to a preparative size column.
Fujifilm Wako offers columns packed with the same packing agent from analytical to preparative size, which enables smooth transfer of preparative conditions.
For example, the following is an example of preparation of Arbutin using Wakopak^® Wakosil^®-II 5C18RS Prep. The Wakopak^® Wakosil^®-II Prep series uses a packing agent with a large particle size (about 5 μm), which allows the column pressure to be kept low even when the flow rate is increased. The columns are available in 4.6 mm to 50 mm I.D., depending on the preparative scale.

Preparative Condition Setting Procedure

1. Determine the preparative conditions with an analytical size column (4.6 mm × 250 mm).

→Select an appropriate packing agent, mobile phase, and other separation conditions on an analytical scale.

2. Set the sample loading volume (injection volume) and flow rate based on the ratio of column cross sectional area between analytical size column and preparative column.

→If the same packing agent can be used for both analytical and preparative columns, equivalent separation can be obtained at approximately the same column pressure by setting the flow rate and the injection volume in proportion to the cross-sectional area of the column.

3. Perform preparation with a preparative column.

Analysis Example of Arubutin (Preparative Example)

Suppose that Arbutin analyzed on a 4.6 mm I.D. Wakopak^® Wakosil^®-II 5C18RS column is scaled up to 20 mm, 33 mm, and 50 mm I.D. columns with the same packing agent, particle size, and column length. It is assumed that almost equivalent chromatograms can be obtained by increasing the flow rate and the injection volume* to match the cross-sectional area ratio. The following chromatograms actually measured show that the peak shape, peak width, and retention time of each component are almost equivalent.
Thus, if a pair of columns with the same packing agent and different I.D. are available, scale-up can be easily performed.

* There are two ways to set the sample loading volume: concentration loading, in which the injection volume is fixed and the sample concentration is increased, and volume loading, in which the sample concentration is fixed and the injection volume is increased. The volume loading method is used in this test.

Peak No.	Component
1	Arbutin

Analysis (Preparative) Conditions

* Cross sectional area ratio when the cross sectional area of a column with an I.D. of 4.6 mm is set to 1.
Cross sectional area = radius × radius × 3.14 (π).

It has the highest number of theoretical plates among our column products.

Click here for information on the analytical columns of the same series (Wakopak^® Wakosil^®-II HG).

Column Information

Silica gel	High purity silica gel with low metal content
Average particle size	about 5 μm
Specific surface area	300 m2/g
Pore size	12 nm
Pore volume	1.00 mL/g
Functional group	Octadecyl group (ODS, C18)
Primary modification	Monomeric
End cap processing	✓
Carbon content	16.0%
USP Code	L1
Column I.D.	4.6 mm, 10 mm, 20 mm, 50 mm
Recommended pH range for use	2-7.5

Toad Venom analysis (preparative)

Peak No.	Component	Peak No.	Component	Peak No.	Component
1	Bufalin	2	Cinobufagin	3	Resibufogenin

Analysis condition

Column size	：	4.6 mm × 250 mm	20 mm × 250 mm	33 mm × 50 mm	50 mm × 250 mm
Flow Rate	：	1.0 mL/min.	18.9 mL/min.	51.5 mL/min.	115.8 mL/min.
Column pressure	：	60 kg/cm2	54 kg/cm2	58 kg/cm2	60 kg/cm2
Injection volume	：	20 μL	380 μL	1.0 mL	2.3 mL
Column	：	Wakopak® Wakosil®-II 5C18HG Prep
Colum temperature	：	R.T.
Eluent	：	A) CH3CN/0.1%CH3COOH=40/60 (v/v) B) CH3CN/0.1%CH3COOH=80/20 (v/v)
Gradient	：	Time (min.) B conc. (%) 0-28 0 28-40 100
Detection	：	UV 300 nm

Sample	：	N) Dibutyl phthalate Rs) Dipropyl phthalate, Dibutyl phthalate
Column	：	Wakopak® Wakosil®-II 5C18HG Prep
Column size	：	4.6 mm × 250 mm, 20 mm × 250 mm, 33 mm × 250 mm, 50 mm × 250 mm
Eluent	：	CH3CN/H2O=65/35 (v/v)

Wakopak^® Wakosil^®- II RS Prep is thoroughly end capped and can be used with aqueous eluents. Therefore, mixed samples with a wide range of polarities and basic compounds can be separated without tailing.
In addition, it has a large specific surface area of 400 m²/g and shows high retention capacity.

Click here for information on the analytical columns of the same series (Wakopak^® Wakosil^®-II RS).

Column Information

Silica gel	High purity silica gel with low metal content
Average particle size	about 5 μm
Specific surface area	400 m2/g
Pore size	12 nm
Pore volume	1.20 mL/g
Functional group	Octadecyl group (ODS, C18)
Primary modification	Monomeric
End cap processing	✓
Carbon content	17.0%
USP Code	L1
Column I.D.	4.6 mm, 10 mm, 20 mm, 50 mm
Recommended pH range for use	2-7.5

Arbutin analysis (preparative)

Peak No.	Component
1	Arbutin

Analysis condition

Column size	：	4.6 mm × 250 mm	20 mm × 250 mm	33 mm × 250 mm	50 mm × 250 mm
Flow Rate	：	0.8 mL/min.	15.1 mL/min.	41.2 mL/min.	92.6 mL/min.
Column pressure	：	45 kg/cm2	46 kg/cm2	49 kg/cm2	44 kg/cm2
Injection volume	：	20 μL	380 μL	1.0 mL	2.3 mL
Column	：	Wakopak® Wakosil®-II 5C18RS Prep
Colum temperature	：	R.T.
Eluent	：	A) CH3COOH/H2O=1/99 (v/v) B) CH3CN/H2O=30/70 (v/v)
Gradient	：	Time (min.) B conc. (%) 0-19 0 19-37 100 37-41 0
Detection	：	UV 280 nm

This column uses a polymeric type ODS packing agent and shows very high durability as the ODS is not easily hydrolyzed even when the mobile phase is acidic (pH 1.4). Due to the bonding properties of polymeric ODS, separation behavior may differ from that of common monomeric ODS silica gel and may exhibit very specific behavior. Therefore, it is useful for the separation of peptides and structural isomers.

Click here for information on the analytical columns of the same series (Wakopak^® Wakosil^®-II AR).

Column Information

Silica gel	High purity silica gel with low metal content
Average particle size	about 5 μm
Specific surface area	300 m2/g
Pore size	12 nm
Pore volume	1.00 mL/g
Functional group	Octadecyl group (ODS, C18)
Primary modification	Polymeric
End cap processing	✓
Carbon content	18.0%
USP Code	L1
Column I.D.	4.6 mm, 10 mm, 20 mm, 50 mm
Recommended pH range for use	1.4-9.4

Acid Resistance Data*

* The above acid and base resistance data were measured using Wakopak^® Wakosil^®-II AR, not using preparative columns.

Standard normal phase preparative column packed with silica gel without functional group modification. In this column, high purity silica gel with low metal content is used as packing agent, making it ideal for the preparative separation of compounds that are affected by metal impurities.
Click here for information on the analytical columns of the same series (Wakopak^® Wakosil^® SIL series).

Column Information

Silica gel	High purity silica gel with low metal content
Average particle size	about 5 μm
Specific surface area	300 m2/g
Pore size	12 nm
Pore volume	1.00 mL/g
Functional group	Silanol group (OH)
Primary modification	-
End cap processing	-
Carbon content	-
USP Code	L3
Column I.D.	4.6 mm, 20 mm, 50 mm
Recommended pH range for use	1.0-4.0

Benzoate esters analysis (preparative)

Peak No.	Component	Peak No.	Component	Peak No.	Component
1	Hexyl Benzoate	2	Propyl Benzoate	3	Methyl Benzoate

Analysis condition

Column size	：	4.6 mm × 250 mm	20 mm × 250 mm	33 mm × 250 mm	50 mm × 250 mm
Flow Rate	：	1.0 mL/min.	18.9 mL/min.	51.5 mL/min.	115.8 mL/min.
Column pressure	：	18 kg/cm2	18 kg/cm2	16 kg/cm2	14 kg/cm2
Injection volume	：	10 μL	190 μL	500 μL	1.15 mL
Column	：	Wakopak® Wakosil®-II 5SIL Prep
Colum temperature	：	R.T.
Eluent	：	n-Hexane/Ethyl Acetate=100/0.5 (v/v)
Detection	：	UV 254 nm