Concept of Size Exclusion Chromatography column

 Size Exclusion Chromatography column

1.1 Theory of SEC mode
The separation modes explained earlier use chemical and/or ionic characteristics of packed gel and analytes. In contrast, SEC is completely different from other separation modes as it using analytes' physical characteristics.

 

Figure 1a	Figure 1b

As explained previously, there are pores on the surface of packed gel. Figure 1a illustrates the SEC separation: The smallest components in the sample mixture (3) cam enter deep into the pore. While larger component (1), cannot enter the pore. The middle-sized component (2) enters the pore in some depth. The components that can enter the pore deeper obviously will spend longer time in the column, thus the elution sequence is biggest to smallest: (1) > (2) > (3). As shown, the SEC separates components according to their sizes. If the diameter of a component is equal to the maximum pore size, like (4) (Figure 1b), it will not be able to enter the pore, and thus will not be retained. For any components bigger than (4), such as (5) and (6), will also not be retained. This means that even they are different in sizes, they will not be separated. The minimum molecular weight (MW) that cannot enter the pore (in this case (4)) is called exclusion limit. In another word, components bigger than the column's exclusion limit cannot be separated by the column. It is important to choose a column that is suitable for the target compound analysis. At Shodex we offer columns packed with wide range of pore-sized gels. The exclusion limit for each column is listed in our catalog. For the best separation, it is recommended to choose a column with smaller exclusion limit, but which covers the MW of the analyte.
MW of a compound is calculated from adding the atomic mass of each component. For example, methylalcohol (CH₃OH) contains four hydrogen (atomic mass 1), one carbon (atomic mass 12), and one oxygen (atomic mass 16).
Thus MW of methylalcohol is 1 x 4 + 12 x 1 + 16 x 1 = 32.

Figure 2

However, it is important to note that MW and the actual molecular size are not always equal when comparing different compounds. As figure 2 shows, even when two compounds have the same MW, one may have less-dense structure while other have very dense structure, so they have different molecular sizes. Even for the same compound, if different solvent was used to dissolve the sample, it can change the molecular structure and consequently it may change its size. The exclusion limit listed in our catalog indicates the standards and conditions used. If the same condition and the sample were to be analyzed, there will not be a concern, but if either condition or sample is different, the listed exclusion limit can be only used as a reference.

1.2 Molecular Distribution Analysis
For the separation modes explained earlier were used to separate each component in a sample. In addition to the separation purpose, SEC is also used to determine the MW distribution of a sample. For an example, polystyrene has a structure as shown below, i.e., it is made of repeating unit of styrene C₆H₅CH=CH₂.

Figure 3

The MW of styrene is 104, thus polystyrene with MW 1,040,000 is made of 10,000 styrene. Although, during the actual production of polystyrene, it is impossible to make compound only with 10,000 styrene. Instead, there will be a variation and the end product will be a mixture of 10,000 styrene unit as well as one with 10,012 styrene units, 9,985 styrene units etc… The mixture will show distribution in its MW with 10,000 as the mode.
In order to obtain a MW distribution information, first it requires a preparation of calibration curve: A set of calibration standards with known MW (Figure 4) is analyzed and then elution time vs. log MW of the calibration standard (Figure 5a) is plotted. The line connecting those plots is the calibration curve. Next step is to analyze the actual sample. In this example, the sample peak started from 13.9 min to 18.8 min. This indicates that the sample contained components with MW 6,200 to 1,100,000 (Figure 5b).

Figure 4	Figure 5

 

MW	9,120,000	1,100,000	400,000	128,000	33,000	6,200	1,350
Elution time	12.3min	13.9min	14.8min	15.8min	17.1min	18.8min	20.0min

Figure 6

It is important to determine the MW distribution of polymers to understand their physical properties. There are also other indexes important for understanding polymers' properties. They include weight average (Mw), number average (Mn), and peak average (Mp). Mp can be obtained from the position of peak top, in the above example, it is ~100,000 Da. However, obtaining the values for Mn and Mw requires a complex calculation. Many of recent software is designed to provide those values automatically.
The actual calibration curve looks like figure 6. Any components with MW larger than exclusion limit will be eluted together. Also there are ranges show linearity and no-linearity. Only the MW range that provides linearity can be applied for the MW determination.

Figure 7

1.3 Separation Based on SEC
In addition to the MW distribution determination, SEC is also used to separate components. In general, the SEC column packed with gels having a large pores (large exclusion limit) is used for the MW distribution determination of polymers, while the one with smaller pores (small exclusion limit) is often used for the separation of small components.
For example, figure 7 shows the separation efficiency comparison of two columns. Three components were to be separated. Each column provides different calibration curves: Column 2 that has smaller slope provides better separation between the three components.
This is true for any other SEC columns; the column with smaller slope calibration curve will provide better separation compared to the one with bigger slope.

1.4 Type of SEC
There are two types of SEC: One uses organic solvent and the other uses aqueous solvent as a mobile phase. The one uses aqueous solvent is called Gel Filtration Chromatography (GFC) or aqueous SEC. The first GFC method was developed by J.Porath and P.Flodin in 1959, in Sweden. They used a cross-linked dextran gel to separate proteins. The columns filled with cross-linked dextran gels are still commercially available, but there are many other types of gels developed and used in biochemistry and pharmaceutical fields.
In contrast, the one uses organic solvent such as tetrahydrofuran (THF) or chloroform is called Gel Permeation Chromatograpny (GPC) or organic SEC. The first GPC method was developed by J.Moore in1964 US. The cross-linked polyethylene gel was used for the MW distribution analysis of synthesized polymers. Even after the big improvement of HPLC, the method used by J.Moore is still a very effective one for the MW distribution analysis of hydrophobic polymers and oligomers.

 

2. Gel Filtration Chromatography (GFC)
GFC columns are available in silica and polymer-base. When comparing the two types, the slope of the silica-column calibration curves is smaller. Thus, the separation efficiency is higher for the silica column. However, very large pore-sized silica gel cannot be prepared due to its structural fragility. Whereas the structure of polymer gel is stronger than silica, it is possible to prepare polymer gels with large pores. In addition, since polymers have stronger chemical stability than silica, it can be used with wider selection of solvents. Silica columns are often used for the separation of protein, glycoprotein, peptide, and nucleic acid. Polymer columns are used for the similar application as silica columns, but can also be used for the MW distribution analysis of aqueous-soluble large polymers.

 

3. Gel Permeation Chromatography (GPC)
For GPC columns, polymer based gel is used. Most frequently used is polystyrene divinylbenzene copolymer. It is important to find a solvent that can completely dissolve the sample. In general, the solvent used to dissolve the sample will be used as a GPC mobile phase. THF is one of the most often used solvents. Other commonly used GPC solvent includes chroloform, dimethylformamide (DMF), hexafluoroisopropanol (HFIP), quinolin, o-dichlorobenzene, tetrachlorobenzene etc… When using a solvent that is different from the shipping solvent, obviously the eluent has to be replaced. The eluent replacement requires careful steps to prevent potential damage on the packed gels.

 

4. Linear Column
The separation efficiency of SEC column increases as the length of column increases (With expense of analysis time and solvent consumption). In order to increase the separation efficiency, it is more common to connect multiple columns in series. For multiple column usage, generally columns with the same type are connected. For an example, 2 to 4 of Shodex GPC KF-806 columns can be connected in series. However, for the analysis of wider MW range sample, different columns are used together. An example is to connect GPC KF-804, KF-803, and KF-802 in series. A problem may arise when connecting different pore-sized columns is the linearity. Even though, each column provides linear calibration curves, when they are connected together, it does not necessary provide a straight calibration curve. Linear columns (mixed-gel columns) are developed in order to solve such a problem. A linear column is packed with a mixture of gels having different pore-sizes. The linear column covers a wider MW range analysis with a straight calibration curve. Thus, instead of using KF-804, KF-803, and KF-802 in series, using 3 x KF-804L will assure providing a straight calibration curve over a wide MW range (the linear columns are named ending with either L or M). Whereas, linear calibration type, LF series, is more recent type that is filled with multi-porous gels, i.e., on the surface of single gel, there are pores with different sizes. This provides even better linearity than linear columns. Figure 8 illustrates the differences between the columns and resulting peaks obtained by each column. Linear types are especially recommended for the MW determination of compound having a wide MW distribution.

 

Figure 8

 

 

5. Downsized Column
It is a very common request that researcher would like to decrease the analysis time. Also considering the environmental issues, it is preferred to reduce the organic solvent usage. By reducing the column size, it can reduce the analysis time as well as solvent consumption. However, by simply reducing the column size, it will also lower the separation efficiency. In order to reduce the column size while keeping the separation efficiency, it is necessary to make the gels smaller. The resulted columns are called downsized columns. Shodex GPC downsized columns were developed in the order of 1 to 4 (Table below). The column volume is decreased and particle size of the gel used was also decreased.

 

Name of the column	Column Size (ID mm x L mm)	Column Volume (mL)	Particle Size (μm)
1. GPC A-800, AC-800, AD-800 etc	8.0 x 500	25.1	10
2. GPC KF-800, K-800, KD-800 etc	8.0 x 300	15.1	6
3. GPC KF-600 etc	6.0 x 150	4.2	3
4. GPC KF-400HQ etc	4.6 x 250	4.2	3

Guidance for column selection is such:
1. GPC A-800, AC-800, AD-800 series are the discontinued items.
2. KF-800, K-800, KD-800, HFIP-800 series are the standard GPC columns.
3. KF-600, K-600, HFIP-600 series are the rapid analysis downsized columns.
4. KF-400HQ series are for the high performance analysis.
Type 3 and 4 require a use of semi-micro type HPLC system in order to provide the desired improvements. Thus, unless such a system is available, standard column (Type 2) is recommended.

 

6. Analysis of Polymers that Require Special Conditions
For the samples that cannot be dissolved in regular GPC solvent such as THF, chloroform, and DMF, there are two methods that are commonly used. They are (1) High temperature GPC method and (2) HFIP solvent method. Their details are explained below.

6.1 High Temperature GPC
Some plastic materials such as polyethylene (PE) and polypropylene (PP) does not dissolve in organic solvents at room temperature. Since they dissolve at elevated temperatures around 140 to 150°C, GPC analysis can be carried at this high temperature conditions. GPC HT-800 series is filed with toluene and suitable for the analysis between 100-150°C. Some analysis requires even higher working temperature, and for those, GPC UT-800 series can be recommended with a use up to 210°C.

6.2 HFIP
As mentioned, some samples require high temperature to be dissolved and analyzed. In order to analyze sample at the elevated temperature, it requires special high-temperature GPC system. A disadvantage apart from its high capital cost, the high-temperature GPC system requires a long warm-up time to stabilize its temperature.
Polyethylene terephthalate (PET) used for clear-plastic bottles, polybutyl tetraphtalate (PBT), and/or nylon have been thought to require a high-temperature GPC system for the analysis. However, it was found that they dissolve in HFIP at room temperature. As the name suggest, GPC HFIP-800 series column is filled with HFIP and suitable for mentioned compound analysis at room temperature. The price of HFIP is much more expensive than other generally used organic solvent, but considering the price of high-temperature GPC system installation, it is not so extreme. Moreover, the solvent can be recycled after distillation. Therefore the running cost of HFIP method is not as expensive as it might be thought.

 

7. Multi-Mode Column
Asahipak GS-HQ series column is a very unique column. Basically, GS-HQ series column is a SEC Column, but more efficient method for the column is to use as a multi-mode column by selecting a certain solvent. What Shodex call muti-mode is a mixed separation mode, providing a combination of two or three of SEC, ion-exchange, and/or partition/adsorption modes. Regular GFC columns may also work under mixed separation mode when mobile phase is not properly optimized. Generally speaking, the conditions providing something other than pure SEC mode is not preferred as it interfere with MW measurement. However, GS-HQ series is designed to provide multi (mixed) separation modes. In some analysis, a desired separation cannot be obtained by single SEC, ion-exchange, and/or partition/adsorption modes, but the multi-mode may solve this problem. A disadvantage of multi-mode is that it is difficult to predict a separation pattern, in other word; there are chances of separation which was not able to be done by any other single separation mode.

 

8. Multi-Solvent Column
Generally GFC column cannot be used with non-polar organic solvents and similarly, GPC column cannot be used with aqueous solvents. If "wrong" solvent was used, it will break the column. However, GF-HQ series column can be used with many different solvent that are used for both GFC and GPC analysis. A column that is usable under such a wide solvent selection is very rare and unique to Shodex.

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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