Symbolic image of drug analysis

Reversed phase chromatography

Columns (with C18) for the analysis of non-polar substances

in drugs, food or biological samples

1. Product overview

2. Tutorial video

3. Theory of reversed phase chromatography

1. Product overview

Shodex Asahipak ODP Series.jpg

Asahipak ODP-50 series

Polymer-based column with C18 functional groups

  • General use for pharmaceuticals and food

  • Relatively large pore size is also suitable for the analysis of amino acids, peptides, and proteins

  • Usable in a wide pH range from pH 2 to 13

  • Usable in 100 % water and buffer solution

  • Best used for the analysis of basic substances

  • Recommended for ELSD, CAD, MS/MS

  • ODP-50 fulfills USP L67 requirements

 

RSpak DE, DM and DS columns

Polymer-based reversed phase columns without C18 groups

RSpak DE-213, DE-413 and DE-613

  • Wide working pH range (from pH 2 to 12), usable in 100 % water and buffer solutions

  • extra small pore size of 25 Å

  • for the analyis of vitamins, preservatives, organic acids

  • Fulfill USP L71 requirements (polymethacrylate particles)

RSpak DM-614

  • Suitable for the analysis of amino acids and water-soluble vitamins

  • pH range from 2 to 10

  • Fulfills USP L39 requirements (polyhydroxymethacrylate particles)

 

RSpak DS-413 and DS-613

  • Suitable for reversed phase analysis of highly hydrophilic substances that are not well retained by ODS columns

  • Fulfill USP L21 requirements (styrene divinylbenzene copolymer particles)

Shodex RSpak DE DS DM Series_square.jpg

RSpak NN and JJ columns

Polymer-based multi mode columns with reversed phase and ion exchange chromatography

RSpak NN-814

  • modified with sulfo groups supports multimode (reversed phase and cation exchange) analysis

  • Ideal for the analysis of complex samples containing neutral and ionic substances

  • For the separation of water-soluble substances such as nucleotides, nucleosides, nucleobases, water-soluble vitamins and amino acids

RSpak JJ-50 2D

  • modified with trace amounts of quaternary ammonium groups, and supports multimode (reversed phase and anion exchange) analysis

  • Ideal for analysis of complex samples containing neutral and ionic substances

ODP2 series

New generation of polymer-based reversed phase columns

  • higher polarity than classical RP column

  • enhances retention of polar/basic substances

  • for small molecules in the presence of protein matrix

  • no silanol-groups -> no adsorption

  • ideal for LC-MS/MS

  • real pH stability

Shodex ODP2 Series.jpg
Shodex Silica C18.jpg

Silica C18 columns

Classic reversed phase columns with C18 groups

C18 and C18M

  • For HPLC

  • Fully end capped ODS column available at very reasonable price

  • Fulfills USP L1 requirements

C18U

  • For UHPLC

  • Achieves high performance analysis with sub-2 µm particles

  • Organic/inorganic silica hybrid particles improved alkali durability (from pH 1 to 12)

  • Usable in 100 % water and buffer solution

  • Fulfills USP L1 requirements

 

2. Tutorial video

 

3. Theory about reversed phase chromatography

1. Reversed-Phase Column

 


1.1 ODS column
The packing material used for reversed-phase column is often made of silica gel modified with functional group. In the story of after work activities (Lesson 1), people may go for a drink by knowing the bar, but if there is a promoter standing in front of the bar, handing out flyers, more people may stop by for a drink. This promoter is the functional group in RP column.

 

The most often used functional group is octadecyl. Octadecyl functional group is a straight carbon chain of 18. Its molecular structure is shown below. It is also often called C18 (C eighteen) column. The name of octadecyl is originated from octa meaning number 8 and deci meaning number 10. A numerous number of this hydrocarbon chain is attached on the surface of silica gel. By looking at the structure written above, it may look like a very long chain, but compare to the size of silica gel, it is actually small. Thus, an infinite number of chains can be modified on one silica gel. More over these chains are modified even inside of the silica-gel pores.


Back to our "famous" story, some people may pick-up the flyer, but others may not pay any attention. Similarly, some compounds may "stopped" by the functional group, but others may not. This defines the eluting timing of each component. By using more scientific words, it can be said "a separation is a result of different affinity between the gel and the components in the sample". This affinity is called "partition" or "adsorption" thus this type of separation is also called partition/adsorption chromatography. Precisely speaking, reversed phase mode is a part of partition/adsorption mode.


The gel made of silica-base and modified with octadecyl functional group is called octadecyl silica (ODS) gel. Also the column packed with ODS gel is called ODS column (or called C18 column). Among the silica-based column available in the market, about 80% of them are ODS columns.


Ideally, entire surface of ODS gel is modified with C18 functional group; however there will be remaining spaces that are not modified. Those part are called "residual silanol" and the presence of residual silanols can influence the separation. Often "end capping" is applied to the gels to immobilize the residual silanol. Almost all ODS columns nowadays are end-capped, however depending on the type of analyte, presence of silanol may provide better separation results.

 


1.2 Other Silica-Based Columns
ODS is most popularly used RP column, however since C18 is a long chain, it may retain compounds too much and consequently results in long analysis time. So in those cases, it is better to use functional group with shorter chain, such as C8, C4, and C3.

C8 : Octyl functional group -CH2CH2CH2CH2CH2CH2CH2CH3 C4 : Butyl functional group -CH2CH2CH2CH3 C3 : Trimethyl functional group -CH2CH2CH3

Also there are silica gels modified with phenyl and cyanopropyl functional groups.

 


1.3 Polymer-Based Columns
As mentioned earlier, ODS (silica base) is the most dominant column for RP column. Although polymer-based columns have also been used. Polymer-based columns have some differences compared to silica-based columns.


• Long column life
In general, polymer gel is chemically more stable than silica gel. It is difficult to predict a column life, since it largely depends on how and under what conditions the column is used. Despite that the column life of silica column is about 3 months whereas it is not surprising to see a polymer column provides stable analysis over 1 year.


• Good repeatability
Gel to gel lot difference of silica gel can be large, whereas that of polymer gel is smaller, since it is relatively easy to control the polymer gel production. From column user’s view point, the analytical performance of a new column is expected to be the same as old column, i.e., negligible level of lot-to-lot difference.


• Usability under alkaline conditions
Silica column cannot be used under alkaline conditions where polymer column can. Some basic samples (often pharmaceutical compounds) require alkaline mobile phase to obtain good separations, thus polymer columns are suitable for such analysis. In another case, columns might be clogged with impurities. Silica columns have very small chance of regeneration, but since polymer column can be cleaned with alkaline solvent, it may be possible to remove the impurities and regenerated.


• Resolution and price
General consensus is that polymer columns compared to silica columns are more expensive and provide less resolution. Therefore even though polymer columns have several advantages over silica columns, former is less often used. The price of the polymer column is becoming lower as the technology has been improved. Also considering the longer column life, from a long term point of view, polymer column is not extremely more expensive than silica columns. The biggest concern will be the resolution. However, again the performance of newer polymer column has been improved and is becoming very comparable to silica columns.


Shodex has strength in development of polymer-based RP columns. The most popular Shodex polymer column is ODP series. As you may guess, the name of ODP came from OctaDecyl Polymer. We believe it is easy to remember, as S (silica) in ODS was simply replaced with P (polymer). The next popular polymer-based RP column is DE-413. The packing gel is made of polymethacrylate. What unique about this gel is that is not modified with any functional group. Instead of using the characteristics of modified functional group, the interaction occurs between the sample and the natural characteristics of polymethacrylate. The question about selection of ODP vs. DE-413 is depends on the sample, you may find many applications on our website to see when ODP/DE-413 is suitable.