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Column Selection
Top of Liquid Chromatography Q&A
Q1
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Q15
Q1
What is the difference between capsulation and conventional end capping?
A1
In the first synthetic process of CAPCELL PAK, silica surface is totally covered with silicone monomers, which are later polymerized to form a polymer film. Therefore, CAPCELL PAK does not need a conventional end-capping procedure.
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Q2
Is silica also polymer-coated?
A2
Silica is not polymer-coated. Only columns named "CAPCELL PAK" are polymer-coated.
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Q3
Which column do you recommend for LC-MS prefocussing?
A3
For LC-MS prefocussing, CAPCELL PAK C18 UG120 is recommended. The UG grade allows the quick elution and removal of polar substances and other impurities because the packing material surface is highly hydrophobic and the surface polarity parameter is small. In terms of prefocussing, CAPCELL PAK C18 AQ is also effective with a mobile phase of 100% water. Both UG and AQ have low column pressures and are easy to use.
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Q4
Is it better to use a guard column?
A4
For a sample containing a lot of impurities adsorbing to a column, a guard column is recommended although this depends on the cleanness of the sample. For the guard column, basically use one which has the same packing material, particle size, and inner diameter.
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Q5
How should a C18 column be selected?
A5
Various factors affect separation in a column. Therefore, Shiseido parameterizes these effects by a simple evaluation method to discuss the characteristics of each column. With ten kinds of standard compounds, Shiseido calculated and evaluated the six parameters, such as hydrophobicity, hydrogen bonding, surface polarity, ion exchangeability, coordination, and planar recognition. The most basic effect of a C18 column is hydrophobic interaction, which depends on the carbon content of the packing materials. As the carbon content of the column increases, the retention becomes longer and the separation is generally enhanced. Since the surface polarity of the packing materials and the presence or absence of silanol groups, also affect separation, however, the selection must be made carefully to suit the purpose of analysis. The number of theoretical plates greatly fluctuates as the measuring instrument, measured compound, measuring conditions, and particle size of packing materials change. Regarding the particle size in particular, the number of theoretical plates and the pressure increase as the particle size becomes smaller. Regarding durability, low operating pressure is preferable. If it is necessary for separation, however, a good number of theoretical plates should be obtained at the sacrifice of pressure. Thus, you should set priority items and select a column according to the purpose of your analysis. For details, please contact us through the Sales representatives in your area ,or Contact shiseido.
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Q6
We are looking for a C18 column of low pressure. Which of your columns is for the lowest pressure?
A6
In general, the CAPCELL PAK Series features a low operating pressure. Of the series, the CAPCELL PAK C18 UG and AQ types feature especially low operating pressures.
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Q7
We want to use a column for a very small sample amount or of a small diameter like a capillary column if possible. Do you offer such columns in your product range?
A7
Shiseido provides columns of 0.3 and 0.5 mm in inner diameter. The standard length is 150 mm.
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Q8
We are analyzing a very hydrophobic compound by using an ODS column. However, the analysis takes a long time and we want to reduce it. What can we do?
A8
Try increasing the organic content. Also, if adjacent peaks are apart enough, it might be a good idea to use a shorter column. Changing the stationary phase type is another strategy. UG type columns show smaller retention for polar analytes, and CAPCELL PAK C8DD is useful to separate highly hydrophobic compounds quickly.
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Q9
We are analyzing basic pharmaceuticals but the peaks tail. Is there a good solution?
A9
The tailing of a basic compound is probably due to the secondary effects of silica packing materials or the influence of silanol groups. Making the mobile phase acidic suppresses this effect. However, this is not easy if many silanol groups remain on the surface of packing materials. If possible, select a column which is not greatly affected by silanol groups. The CAPCELL PAK Series columns are made of polymer-coated silica and so are hardly influenced by silanol groups. Although the retention behavior changes greatly, the ion-pair method also allows analysis without being affected by silanol groups.
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Q10
Which column is suitable for protein analysis?
A10
If the pore size is as small as 12 nm, protein of a high molecular weight (10000 or more) may be adsorbed. Therefore, it is recommended to use a 30 nm column. If the pore size is 30 nm, C1, C8, or C18 can be used for separation. For durability and recovery rate, the C8 SG300 is recommended. The durability tends to improve as the alkyl chain becomes longer. The recovery rate tends to improve as the alkyl chain becomes shorter. Protein of a low molecular weight can be analyzed with a 12 nm column with no problem. For use under acidic conditions including TFA, the CAPCELL PAK C18 ACR is recommended due to its exceptional acid resistance.
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Q11
How can I analyze protein quickly?
A11
For quick analysis, the column is generally made short. If the molecular weight exceeds 10000 in protein analysis, the CAPCELL PAK C8 SG300 is recommended first. With this column, 0 to 100% gradient analysis is performed with formic acid added water and acetonitrile if the detector is MS and TFA added water and acetonitrile if the detector is UV. For protein having a comparatively low molecular weight below 10000, the CAPCELL PAK C8 UG120 or C18ACR or MG is recommended. Since the protein is analyzed by gradient elution, the analysis speed is limited by nature and there are no columns for general HTS analysis (combinatorial chemistry, etc.) at present.
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Q12
What is the difference of protein recovery rate between functional groups?
A12
In the CAPCELL PAK, the packing material surface is coated with polymer to minimize adsorption by the secondary effect of silica gel (influence of metallic impurities or silanol groups). Therefore, there is no remarkable difference in recovery rate attributable to the difference of functional group. Generally, however, the recovery rate decreases but the durability improves as the alkyl chain becomes longer, and the recovery rate increases but the durability deteriorates as the alkyl chain becomes shorter. The CAPCELL PAK has generally improved durability by polymer coating but the influence of adsorption (e.g. hydrophobic interaction by carbon chain) other than the secondary effect of silica is considered to be equal to those on other columns. To improve the recovery rate, select a column of short alkyl chain.
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Q13
Why does the CAPCELL PAK MF have a limit on organic solvent? Can the column be used in the same way as an ordinary reversed phase column if not for a biological sample?
A13
Organic solvent is limited to prevent protein degeneration. The CAPCELL PAK MF as a stationary phase is stable under any commonly used mobile phases. Regarding separation, however, the retention is predicted to be smaller than that of a general phenyl column because hydrophilic groups are introduced.
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Q14
What is the NH2 separation mechanism?
A14
This has the combined separation mode of normal-phase distribution (hydrophilic interaction) and ion exchange. For a neutral substance, the retention increases as the CH3CN content in a mobile phase increases. A basic substance has ionic repulsion with the stationary phase. As the CH3CN content in the mobile phase increases, the ionic repulsion is weakened and the retention increases under the influence of normal-phase partition. Since an increase of salt concentration or an increase of pH weakens the ionic repulsion, the retention increases. For acidic compounds the NH2 phase behaves as a weak anion exchanger. Retention increases (decreases) when salt concentration and pH are lowered (raised).
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Q15
Are there any advantages in replacing NH2 columns of other manufacturers with CAPCELL PAK NH2?
A15
The CAPCELL PAK NH2 produces a chromatogram equivalent of one from the conventional NH2 column if the CH3CN content in the mobile phase conditions is increased by about 5%. The life becomes three or four times longer.
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