Lesson 5: Other Separation Modes, Preparative and Guard Columns

1. Ion Exchange Column

The N and S ends on the magnet attract each other while N and N or S and S ends will repeal each other. The similar phenomenon occurs for electronics: Positive charge and negative charge attract each other while positive and positive or negative and negative charges repeal each other. When compounds dissociate and become ions, they will have electronic charges. Positive ion is called cation and negative ion is called anion. Ion exchange mode uses ionic attraction and repulsion forces. What is different from magnet is that N end of magnet is always N, but molecular ion such as protein can be an anion or a cation depending on the surrounding conditions (mobile phase). By changing the electronic characteristics of the mobile phase, the electronic charge of each component in the sample also changes. They may change from cation to anion (or vice versa) and this creates the interaction between the component and the packed gel. One time it may cause attraction and other time it may cause repulsion. Since the components that experience repulsion will be eluted out (and attraction causes retention) from the column, each component is separated based on their ionic strengths.

It is common to change the mobile phase during an analysis for ion exchange mode. Such method (changing mobile phase) is called gradient method. Most frequent method used for gradient elution is to prepare two mobile phases and change the ratio of two solvents over a time. Contrast to gradient method, the method uses single solvent throughout the run is called isocratic method.

Packed gel for ion exchange column is modified with either anion or cation functional groups. Commonly used functional groups are followings.

Quaternary ammonium (QA): Strong anion exchanger

Diethyl aminoethyl (DEAE): Weak anion exchanger

Sulfopropyl (SP): Strong cation exchanger

Carboxylmethyl (CM): Weak cation exchanger

Gels generally used are porous gel (having pores on the surface). However for the ion-exchange gel, small-sized gel (2.0-2.5 um) without pores is sometimes used. Gels without pores are called non-porous gel and it is effective for fast analysis. Both polymer-based and silica-based columns are used for ion-exchange columns. Though unlike RP columns that mainly uses silica base, ion-exchange columns sometimes require a use of alkali conditions. Thus importance of polymer-based materials is higher with ion-exchange columns than RP columns. Ion exchange is frequently used in biochemistry area such as separation of protein, peptide, and nucleic acids.

 

2. Ion Chromatography Column

There are two types of Ion Chromatography methods: The suppressor method and non-suppressor method. Shodex carries columns suitable for both types. As the name tells, the suppressor method uses a suppressor which removes ions that interfere with analytes measurement. The suppressor system is expensive, but some say the sensitivity of suppressor method is superior to non-suppressor method. In contrast, system required for non-suppressor method is cheaper, but the useable solvent is limited to the ones with low conductivity such as phthalic acid.

 

 

2.1 Suppressor Type Ion Chromatography Column

Shodex IC SI-90 and SI-50 are the suppressor type anion chromatography columns. SI-50 is the higher performance type of SI-90. SI-52 is another suppressor type column suitable for the separation of oxyhalides.

 

2.2 Non-Suppressor Type Ion Chromatography Column

Shodex IC I-524A and NI-424 are the non-suppressor type anion chromatography columns. NI-424 is the higher performance type of I-524A. Shodex IC YK-421 and YS-50 are the non-suppressor type cation chromatography columns. YS-50 is the higher performance type of YK-421. They can separate both monovalent and divalent cations simultaneously.

 

2.3 Other Columns

There are other columns available for specific applications, such as for separation of transition metal and rare earth metal ions.

 

3. Ion Exclusion Column

Compared to ion-exchange column that uses attractive force between anion and cation, ion-exclusion mode uses repulsive force between the anion of analyte and packed gel. Ion-exclusion mode is not use solely: Generally it is a fine balance used with other separation modes such as partition/adsorption mode. For an example, partition/adsorption mode will work to retain the analyte while ion-exclusion will try to exclude analytes. Ion-exclusion mode is often used for organic acid analysis.

 

4. Ligand Exchange Column

The packed gel used for a ligand-exchange column is modified with substances with ionic functional group. The modifier contains metal cation, which is called “counter ion”. The ligand exchange mode is often used for the separation of saccharides using the interaction between positive charge of metal cation and negative charge of hydroxyl group (OH) on saccharide. The number of OH as well as their configurations influences the strength of interaction. Counter ions used include hydrogen, calcium, lead, zinc, and sodium. Often the separation works under combinations of size exclusion and ligand exchange, partition/adsorption and ligand exchange, and/or ion exclusion and ligand exchange modes.

 

5. Affinity Column

The separation mode of affinity column is different from other separation modes explained earlier. The packed gel is modified with so called ligands – similar to the functional groups. The ligand captures only a specific compound. It is similar to key and lock: Among many keys, only one specific key can open the lock. When sample containing mixed components is injected into an affinity column, only a specific component is retained by the ligand and all other components are going to be eluted. By changing the mobile phase, trapped component can be removed from the ligand and eluted. Figure below illustrates the affinity mode separation.

 

 

6. Chiral Column

Frequently compounds used for pharmaceutical material production has optical isomers. Optical isomerism is one type of stereoisomerism that two molecules have the same arrangement of atoms, but they have different orientations in space. One has a mirror image of the other and cannot be superimposed. Another familiar example is right and left hands. They have same shape, but cannot be superimposed. The asymmetric molecule is called chiral. When a beam of light is emitted to the pair of chiral compounds, one rotates the light to clockwise (dextrorotatory isomer, designated d or +) and the other rotates into counterclockwise (levorotatory isomer, designated l or -).

One famous story of chiral compound is thalidomide incident in late 1950s. Thalidomide was sold as a sedative drug and was thought that it does not give much side effect to pregnancy, thus was taken frequently by pregnant women. However it resulted in many severe birth defects. This is due to the chiral compounds; one has sedative effect while other is a potent teratogen. Chiral pairs are very alike but once they are taken by human (animals), their biological activities may be totally different from each other. A problem is that it is difficult to produce only one type of chiral compound during the production of pharmaceutical materials. As mentioned, leaving non-target chiral compound may cause another incident as thalidomide, and thus it is important to separate chiral compounds.

When separating two components by HPLC, the larger the difference in components’ characteristics, the easier the separation. Thus, since chiral pair is very similar, it is extremely difficult to separate. Shodex provides optical isomer separation columns using different ligands: alpha-, beta-, and gamma- cyclodetrin derivatives, L-amino acids derivatives, and bovine serum albumin ligands. Each works under different mechanisms. For example, one uses conformational compatibility differences while other uses metal complex formation capacity differences of the isomers.

 

7. Special Use Columns

There are some columns that are designed for specific applications. Details of each are explained below.

 

7.1 Pesticides (Used at Golf Course) Analysis Column

At golf courses, a large amount of pesticides are used to maintain the field. The pesticides are washed by rain and enter into nearby rivers. The impact to drinking water is a concern. The GOLF-413 is the column suitable for the separation of main pesticides often used at golf courses.

 

7.2 Pesticides (N-Methylcarbamate) Analysis Column

Commonly used HPLC method for the analysis of N-Methylcarbamates is a gradient method. A disadvantage of gradient method is the equilibration time between each analysis. RSpak CARB-413 is designed for the separation of N-Methylcarbomates under isocratic method, reducing the analysis time largely.

 

7.3 Surfactant Removal Column

MSpak PK and MSpak GF are designed to remove surfactants that interfere with protein analysis. This is a cartridge type column which is placed in front of an analytical column, effectively removes surfactant on-line.

 

7.4 Amino Acid Analysis Column

CXpak P-421S is suitable for the separation of 17 essential amino acids in one analysis.

 

7.5 Cyan Analysis Column

Cyan could be present in industrial wastewater and it is toxic. KC-811 6E is suitable for the low detection limit measurement of cyan in water.

 

7.6 Mannitol Analysis Column

USPak MN-431 is assigned under United States Pharmacopeia Column List L19 and 22, suitable for the analysis of mannitol.

 

7.7 Umami Components Analysis Column

Asahipak GS-320 7E is suitable for the separation of nucleic acids components for taste (umami) i.e. IMP, GMP and AMP (nucleotide), and all of their metabolites with simple isocratic elution. It is also used for the food freshness testing.

 

7.8 Sample Clean-up Column

A large amount and variety of pesticides is in use. Some may be found on/in food and they are potential poison for human consumption. Thus, it is important to measure residual pesticide in food. Generally the amount of residual pesticide is very small compared to other components and this makes quantification difficult. Thus it requires removing any other components as a pretreatment. CLNpak EV series are used for this purpose.

 

8. Preparative Column

The columns mentioned so far were “analytical columns” with column internal diameter (ID) of 4.6 ~ 8.0 mm. Purpose of using preparative column is to collect target substances after separating it from other substances. Analysis column can separate these substances, but treating amount per column is not large. On the other hand, preparative columns, larger diameter than analytical columns, can increase treating amount per column. Generally large particle size of packing materials is applied for preparative column, but there are cases that the same packing materials may be used. There are mainly two reasons for collecting components: (1) to use in other analysis and (2) to use for industrial products.

(1)  By using HPLC separation, chromatogram obtained shows the peaks corresponding to the components in the sample. For unknown analyte, the position of peak does not tell any information about the composition of the peak. Thus it requires to collect the unknown peak (analyte) and by using other technique to find out the composition of the analyte. Often mass spectrometer (MS) is used for the identification. Coupling HPLC and MS online is becoming common and the unit system is also commercially available. Also in some research, a certain components of the sample may be required to be used in next experimental sample. By using a preparative column, the specific component can be obtained.

(2)  Use in a large scale collection and/or purification
For preparation of certain products (often for the production of pharmaceutical products), it requires to separate and collect a certain components of raw material in a large scale. The preparative column will be used as a production tool.

For (1), the amount collected will not be so large, thus 20 mm ID preparative column is used. If concentration of target compound in the sample is relatively high, smaller ID column might be sufficient for the purpose. For (2), use in production, the larger ID column such as 50, 100, or 200 mm is generally used.

 

9. Guard Column

The presence of impurities in the sample may cause alternation of column by being deposited inside the column permanently. To prevent such problems, it is necessary to pre-treat the sample by centrifugion and/or filtration. However, there are components that cannot be removed by centrifugion/filtration. If those samples were injected, it may break the column. The guard column is effective preventing the analytical column from being alternated by those impurities. The guard column is a smaller version of the analytical column; packed with the same type of gels as that is in analytical column. The guard column is placed before the analytical column, thus any potential impurities that may cause damages is trapped inside the guard column before it reaches the analytical column. The price of a guard column is generally about one third to one tenth of the analytical column. By using the guard column, it can enhance the analytical column life and thus can be more economical. Although for the relatively low-priced analytical columns, the price of guard column compared to the analytical column could be high. Thus for those columns, the corresponding guard column may not be available. Moreover, the use of guard column is more effective for the polymer-based columns that have expected long column lives compared to silica-based columns.