Reversed phase Column Theory

 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.

 

2. Normal-Phase Column
It is rather complicated to explain detailed-theoretical differences between RP and normal phase, thus we will keep it simple here as an introduction. Gels and mobile phases used for HPLC analysis have different polarities. Water and oil is a famous example of something does not mix: Water is categorized as something with high polarity while oil is categorized as something with low polarity. Oil is a type of carbohydrate, made of carbon and hydrogen; such compound has low polarity. In contrast, water is made of oxygen and hydrogen; such compound has higher polarity. Silica gel without modification has high polarity, but when C18 functional group is modified, the polarity becomes low. RP mode uses gel with low polarity (e.g., ODS) and mobile phase with high polarity (e.g., water, acetonitrile). Normal-phase mode uses gel with high polarity (e.g. silica) and mobile phase with low polarity (e.g. hexane, chloroform). Instead of using the word low or high polarity, it is also common to use words, hydrophilic or hydrophobic. Something easy to dissolve in water (i.e., high polarity) is called hydrophilic and something easy to dissolve in oil (i.e., low polarity) is called hydrophobic.
At the very early stage of HPLC development, silica gel without any functional group was only used. Thus, historically normal-phase mode was developed first and so named "Normal". Then the separation mode which uses opposite separation theory to normal phase was developed and named "Reversed". RP mode is much more popularly used than normal phase nowadays, but we cannot change the historical background, and thus they are still called normal and reversed-phase modes.

 

3. Hydrophilic Interaction Chromatography (HILIC) Column
HILIC is a relatively new concept as a member of partition chromatography. It is considered as a part of normal phase because of its high polarity on the gel surface. The base material can be either silica or polymer and they may be modified with different types of polar functionalities such as amide, amino, diol, and cyano. Compared to normal mode, the mobile phase used for HILIC is very similar to RP mode mobile phase such as mixture of water and acetonitrile. From practical view point, HILIC sits somehow between the RP and normal mode separation. Hydrophilic compounds that were “too polar” to be retained by RP can be analyzed by HILIC using the mobile phase similar to RP condition. Because of this feature HILIC is popularly used for the separation of carbohydrates, especially saccharide which is hydrophilic.
Shodex carries a polymer based amino column, named Asahipak NH2P series. NH2 indicates "amino functional group" and P again stands for polymer. This column is filled with a polyvinyl alcohol based gel, modified with polyamine. As for RP columns, polymer base compared to silica-base column will provide longer column life, durability in alkaline condition, and good repeatability.

 

4. Summary of Shodex Partition/Adsorption Columns
Figure 1 is a summary of Shodex partition/adsorption type columns. X-axis is the polarity of gel; column on the right side is lower polar and left side is higher polar. i.e., the column on right side is used for RP mode and left side is for the normal phase mode. Y-axis is the pore size of the gel. For the analysis of large components, gels with larger pore-size should be selected and vice versa.

HPLC COLUMN SEPARATION THEORY

 Concept  of LC Column Separation

As mentioned in Lesson 1, the actual separation occurs inside the LC column. You may be wondering what is happening inside the column. Let's use "after-work activity" as an example to explain the column separation.

Figure 1. Components of HPLC system

One evening at 6pm, three people, a researcher, a business man, and an office worker were leaving their work. The office worker goes straight home, and arrives home at 7pm. A business man went to a restaurant with his coworker and had a wine. They decided to go to a bar to have few drinks after eating. Then the business man went home at 9pm. The researcher went to a bar after work and had a beer, met someone who also likes drinking. They moved to another bar next door had few more beer. The researcher met his friends at the bar and they decided to go to a Karaoke bar and had another drink. Everyone else went home, but the researcher stopped by at a nearby bar. He drunk till the bar closed and finally went home at midnight. From the view point of home arriving time, we can tell who likes drinking or not: Non-drinker office worker, social-drinker business man, and heavy-drinker researcher. So how this story is relevant to the LC column separation?
The LC column is filled with very small particles (also called gels), and the size of gel is 3 to 15μm (μm=1/1000mm) or smaller. This gel has various "traps". Each sample component has different "characteristics" and interacts with the "trap" differently, i.e., each component may stay inside the column for different lengths. Thus using this time differences, the components are separated.
If we were to go back to the after-work activity story, the bars are the traps and each person's drinking taste is the characteristics. Because of this difference in their characteristics, the "type separation" was possible. However, by changing the conditions, the separation can be changed. For example, the office worker may have gone to a restaurant to have a glass of wine if he was meeting his friends. Or the researcher may have gone home if he had to attend a conference next day. The condition change, in case of LC, is to change compositions of mobile phase, pH, column temperatures etc… By changing the conditions, the components which were not able to be separated may be separated or could be vice versa. This is the difficult point of LC and where the user needs to pay a big attention. In some cases the same "trap" with different conditions may make the separation possible, but in other cases, the same "trap" does not work at any conditions you try. In the latter case, different kind of trap may be required. In "LC word", the former means that using the same column with different conditions (mobile phase/ temperature etc...) and the latter case means to change the LC column. There are different LC column packing materials (base material) available. Moreover using different modifications (addition of chemical compounds on the surface of the gel), a wide variety of LC column can be prepared.
Shodex carries about 1000 different LC columns. In other words, we need to choose a best fit column among these 1000 LC columns.

2. HPLC Separation
Ideally, obtained LC separation result should provide a symmetrical peak shape (Figure 2a). When there is a problem, the peak will not be a symmetrical one and may show leading (Figure 2b) or tailing (Figure 2c). The high-performance column will provide narrower peak (Figure 3a) and low-performance column will provide wider peak (Figure 3b). To measure the performance of the column, we use "theoretical plate number (TPN)". It can be said that the bigger the TPN, the better the column. TPN is directly proportional to the column length, i.e., if the column length was doubled or two columns were used in a series, the TPN is also doubled. When comparing the Shodex column with other company’s TPN, we need to make sure if those column lengths are the same. The Shodex states TPN per column, but some other manufacture states TPN per meter, thus need to pay an attention on the units. The internal diameter of the column also influences the TPN, but it is not as significant as that of column length. Also TPN may differ if different LC settings or measurement methods were used, even using the same column and the same mobile phase.
For the separation of two components in the sample, it is ideal to have separation at the baseline level (Figure 4a). When two peaks are too close, they may overwrap, and results in insufficient separation (Figure 4b-c). The column with higher TPN provides sharper peaks, thus the possibility of overwrapping is smaller than the column with lower TPN.

(a) Normal peak (b) Leading (c) Tailing	(a) High TPN (b) Low TPN	(a) Baseline separation (b) Some peak overwrapping (c) Poor separation
Figure 2. Examples of peak shapes.	Figure 3. Peak shapes and column TPN.	Figure 4. Separation of two components.

Even when peak overwrapping was observed, this may be solved by changing the analytical conditions (e.g. changing mobile phase). If the separation cannot be improved by the conditional changes, different types of LC column may be used.
For the qualification analysis (to identify what components are present in the sample), peak overwrapping may not be a big concern. However, for the quantification analysis (to measure how much each sample is present in the sample), the baseline separation is required for the precise measurement.

3. Types of packed gels
3.1 Silica gel
Silica gel is the most popularly used packing material. Silica, silicon dioxide, has the chemical formula of SiO₂. You may see a small paper bag of silica in food packages stated 'do not eat'. It is used as a dehydrator. The ones used for dehydrator has a gel diameter of 1 mm or larger, but the ones packed in LC columns are very small; few um sizes. There are two types of silica gels. The one has spherical shapes, the other has irregular shapes. Unlike past, the spherical shaped gels are most widely used these days. The silica gel used in LC has pores on the surface of the gel. By having the pores, it provides larger surface area compared to the ones without pores. The size of pore is very small and expressed in angstrom (Å) unit. The silica with pores is called porous silica.
There are few indexes used to express silica gel grades.
Shape : Most silica columns used nowadays contain spherical type.
Size : Smaller size particles have been developed. Current major line is 5μm, but even smaller size 1.5 to 3μm gel is also in use. The smaller gels are packed in smaller column housing and thus decreases the analytical time.
Pore size : There is not a simple good/bad indicator for pore sizes. The right pore size should be determined depending on the size of target analyte.
Surface area : This is the relative surface area of the gel. The smaller the particle size, the relative surface area becomes larger. Also the larger the number of pores, the larger the relative surface area. If all the other indexes are the same, the better performance can be expected from the larger surfaced-area gel. One gram of conventional silica gel provides a surface area of softball field.

3.2 Polymer gel
In the earlier stage of HPLC development, almost always silica gels were used. However, polymer-based column is becoming popular. The generally known polymers include polyethylene and poly propylene. Shodex columns use several different types of polymers as listed below.
(1) Polystylene (Styrene divinylbenzene copolymer)
(2) Polymethacrylate
(3) Polyhydroxymethacrylate
(4) Polyvinyl alcohol
Similar to the silica gel, the polymer gel is manufactured into very small particles.

3.3 Other gel
Other than silica and polymer gels, the gels used include natural substances such as cellulose, agarose, dextrin, and chitosan, and members of ceramics such as hydroxyapatite and zirconia. However, their use is very limited.

4. Types of separation mode
As explained earlier, there are many different types of "traps" in the column, and depending on the "trap" there are different types of columns. This "trap" is called separation mode. Generally used separation modes in LC are listed below.
(1) Reversed-phase (RP) mode
(2) Normal-phase (NP) mode
(3) Hydrophilic Interaction (HILIC) mode
(4) Ion exchange (IE) mode
(5) Ligand exchange mode
(6) Ion exclusion mode
(7) GPC mode
(8) GFC mode
(9) Multi mode
(10) Affinity mode
(11) Chiral mode

 Normal phase

Normal phase HPLC systems are similar to the flash-column chromatography that you might be familiar with.

A silica stationary phase is eluted with a non-polar solvent such as hexane, or a fairly non-polar solvent mixture such as 2-propanol in hexanes. In normal phase chromatography, only organic solvents are used.

In the normal phase, polar molecules elute slowly, and non-polar (greasy) molecules elute quickly.

Reverse phase

Reverse phase is essentially the opposite of normal-phase.

A non polar stationary phase (often silica in which the free hydroxyl groups are end-capped with something greasy, C18 chains are common but many many variants are possible) is eluted with a polar solvent such as acetonitrile/methanol, or a fairly polar solvent mixture (acetonitrile water mixtures are common, or methanol water mixtures).

In the reverse phase, polar molecules elute quickly, and non-polar (greasy) molecules elute slowly.

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