Technical Support
Feature of columns / materials
We have 15 different kinds of C18 columns. They are divided in 4 groups, Hybrid silica based YMC-Triart columns, Core-shell type silica based Meteoric Core columns, Silica-based columns and Polymer-based YMC-Pack PolymerC18.
YMC-Triart columns exhibit extraordinary stability, low operating pressure, and excellent performance. Meteoric Core columns provide outstanding resolution compared to fully porous silica based columns.
To break down further within the Silica-based columns, they are divided in 3 groups, Pro series, YMC-Pack series (excl. PolymerC18) and J’sphere series. Pro series features in a superior performance and excellent reproducibility. Pro series with an efficient endcapping technology is superior to YMC-Pack ODS series and J’sphere series. You can find more information in the following C18 column selection guide.
ODS-A and AM are conventional ODS. ODS-AQ provides a lower rate of carbon content and is suitable for separation of hydrophilic compounds. ODS-A and -AM have the same basic physical properties such as the base material, the rate of carbon content and the separation characteristics, however -AM is produced under more strict Standards of Quality Control.
Polyamine II and PA-G are chemically bonded with polyamine where NH2 is with aminopropyl group. Polyamine II and PA-G are superior to NH2 in durability. They also have difference in selectivity. Polyamine II and PA-G have different ligand structure of polyamine. Polyamine II is superior to PA-G in durability.
General ODS (C18) packing material is a silica gel bonded with octadecyl groups. This is in the result of a reaction between silanol groups and octadecyl groups on the silica surface. However some silanol groups remain after the reaction. It is impossible for all the silanol groups to react because of steric hindrance of octadecyl groups. Such residual silanol groups create a secondary interaction in chromatography, which, in many cases, affects on chromatograms by, in general, causing a peak tailing of basic compounds or irreversible absorption to the column. Therefore, a secondary silanization on residual silanol groups with unbulky silanization reagents should be performed. This process is called “endcapping”. Trimethylsilane (TMS) is commonly used in “endcapping” process.
“Triart C18”, “Hydrosphere C18” and “ODS-AQ” columns can be used with 100% aqueous mobile phase. On conventional ODS columns, retention time is shortened due to the incompatibility between water and material surface with high hydrophobicity. Water tends to be expelled from the pores on material. The retention time hardly shortened on “YMC-Triart C18”, “Hydrosphere C18” and “ODS-AQ” because they are capable of solvation between mobile phase and hydrophilic surface by reducing the density of C18 functional groups.
Column handling
Applicable pH range and temperature
Column Type | pH range | Usable temperature range | |
---|---|---|---|
Regular use | Upper limit | ||
Triart C18, C18 ExRS, C8 | 1 – 12 | 20-40℃ | pH 1-7 : 90℃ pH 7-12 : 50℃ |
Triart Phenyl | 1 – 10 | 20-40℃ | 50℃ |
Triart PFP | 1 – 8 | 20-40℃ | 50℃ |
Triart Bio C18 | 1 – 12 | 20-40℃ | pH 1-9 : 90℃ pH 9-12 : 50℃ |
Triart Bio C4 | 1 – 10 | 20-40℃ | pH 1-7 : 90℃ pH 7-10 : 50℃ |
Pro C18, Hydrosphere C18 | 2 – 8 | 20-40℃ | 50℃ |
Pro C18 RS | 1 – 10 | 20-40℃ | 50℃ |
J’sphere ODS-H80 | 1 – 9 | 20-40℃ | 50℃ |
PolymerC18 | 2 – 13 | 25-35℃ | 65℃ |
Triart Diol-HILIC | 2 – 10 | 20-40℃ | 50℃ |
Reversed-phase (Other than mentioned above) Normal phase (SIL, Polyamine II) | 2 – 7 | 20-40℃ | 50℃ |
*The given data is subject to change depends on product types. Those data should be confirmed with Instruction manual when the column is used.
When columns are not used for a long time, keep them in a cool place after replacing with the shipping solvent as indicated in the attached inspection report. Do not keep the column in the mobile phase with salt or acid regardless of whether or not it is in a short period of time. Close the airtight stopper tightly to prevent the solvent from volatilizing.
Perform an inspection test under the same conditions as the inspection report attached to the column at the time of purchase. Columns are evaluated to be effective and have no change in performance if the result indicates no irregularity in retention time, theoretical plate number, peak asymmetry, etc. Columns which indicate no irregularity in the said criteria after using several years from purchase, however, may have changes in separation characteristics for compounds such as ionic compounds.
It is advisable to avoid using them for method development. Reproducibility may not be obtained with new columns.
Triart Series: Acetonitrile (100)
Pro Series, ODS-A, AM, AQ, etc.: Acetonitrile/Water (60/40)
J’sphere Series: Acetonitrile (100)
* Indicated in the COLUMN INSPECTION REPORT.
- Remove highly hydrophobic substances adsorbed onto the gel
- Use solvent with a stronger elution ability than mobile phase. For example, cleaning reversed-phase columns, use solvent with increased ratio of organic modifier and flush the volume of 10 times as much as the column volume.
- Renaturation of gel surface condition
- Irregularities are observed in Peak asymmetry and retention time.
In silica-based packing material, separation behavior may be affected by the conditions of residual silanol groups whether in dissociation or in non-dissociation. Washing with acidic solvents may be effective in such case. Washing with a mixed solvent of 0.1% aqueous phosphoric acid solution and organic solvent* can perform the renaturation of silanol groups to the dissociation state.
* Ratio of organic solvent: 10 to 60%.
- Irregularities are observed in Peak asymmetry and retention time.
To analyze samples contain a lot of contaminants, guard column is effective and can improve the durability of main columns. We recommend guard columns with the same packing materials as main columns. Guard columns with different material may cause defects in peak asymmetries and reproducibility. We have them in 2 types, conventional type and cartridge type. We recommend cartridge type if guard columns require a frequent replacement. Inner diameter should be same as the main column or smaller.
“WT” indicates Waters connector compatible, “PT”, “PTH”, “PTC” or “PTP” indicates Parker connector compatible. The majority of columns in the market are of these types. There are several connection types other than Waters and Parker compatible types such as Shimadzu, JASCO, Hitachi, etc. The difference in these connection types is the length of tubing section coming out from the tip of ferrule. The connector types of column and tubing system should be the same, or tubing and column may fail to fit well and cause leakage and defects in peak asymmetry. If your system has something other than Waters, a connection adapter or a ferrule replacement may be required. * PEEK inch screw thread built-in ferrule would not have the problem.
The end of the product number | *Port depth | Style of endfitting |
PT/ PTH/ PTC/PTP | 2 mm | Parker style (UPLC compatible) |
WT/ WX/ WTG/WP/WTS | 3 mm | Waters (W) style |
UPLC is a registerd trademark of Waters Corporation
Flow rate on Semi-micro column (hereafter columns in 1.0 to 2.0 mm inner diameters will be mentioned as semi-micro columns) is 50 to 200 µL/min in general. It can be increased if the length of column is short and back pressure is low. Commonly used HPLC System is applicable, however, with pumps, flow cell of detectors and tubing system designed for semi-micro column is more suitable.
- Step 1
- Determine separation conditions by using analytical columns.
- Step 2
- Study the preparative scale. Set the particle size of the packing material and the inner diameter of column in consideration of the sample volume.
- Step 3
- Optimize the separation conditions by using analytical columns with inner diameter of 4.6 mm or 6.0 mm packed with the selected packing material. If the particle size of the packing material is the same as in the Step 1, this process can be omitted. If the preparative scale is more than 100 mm in inner diameter, another process using a column in a diameter of 20 mm will follow to determine the loadability and calculate the running cost.
- Step 4
- Proceed with the preparative separation.
Solution of column trouble
Wash the column under the method in “How to clean the columns? 1. Remove highly hydrophobic substances adsorbed onto the gel” Reduce the flow rate accordingly in order to keep the column pressure adequate when flashing the column. If the cause is believed to clog frit or terrible contamination, washing by reversed direction flow will be very effective.
* If pressure increase is observed often after washing the column, take such measures as sample pretreatment or using guard columns to prevent the problem
Trouble Shooting tips : If Column Pressure Increases
- Inappropriate Mobile phase
- In the case of ionic analyte if pKa of the analyte and pH of mobile phase are close, it causes defects in the peak shape.
Set the pH of mobile phase distant from pKa.
- In the case of ionic analyte if pKa of the analyte and pH of mobile phase are close, it causes defects in the peak shape.
- Influence by dissolving solvent of sample
- If dissolving solvent of sample and mobile phase are not the same, it causes defects in the peak shape. Dilute the sample solution with mobile phase or reduce the injection volume.
- Overloading sample injection
- Overload will cause defects in the peak shape.
Reduce injection volume.
- Overload will cause defects in the peak shape.
- Insufficient equilibration time
- When variance in pH is wide between the current and previous mobile phase or the buffer concentration of mobile phase is low, column equilibration may take a while.
- Column contamination and degradation
- In the case of contamination, wash the column according to “How to clean the columns? 1. Remove highly hydrophobic substances adsorbed onto the gel”. If column is in degradation, it is impossible to regenerate. The column should be replaced.
- System problem
- Dispersion may occur within tubing between injector and column or the flow cell of detector which results in peak tailing and/or broading. System should comply with semi-micro use.
Trouble Shooting tips : Peak shape anomaly
Following solutions are introduced depends on causes.
- Injector fouling (carry-over)
- If the ghost peak appears when injecting mobile phase only, wash the injector.
- Gradient Analysis
- When hydrophobic impurities are eluted by a stronger solvent, it appears as a ghost peak. Clean the column according to “How to clean the columns? 1. Remove highly hydrophobic substances adsorbed onto the gel”. If you still can’t eliminate them, the cause should be impurities of solvent.
Use a higher grade solvent. Trap the impurities by attaching guard column between the solvent delivery pump and the mixing chamber.
- When hydrophobic impurities are eluted by a stronger solvent, it appears as a ghost peak. Clean the column according to “How to clean the columns? 1. Remove highly hydrophobic substances adsorbed onto the gel”. If you still can’t eliminate them, the cause should be impurities of solvent.
Trouble Shooting tips : The Cause of the Ghost Peak
Flush the column with solvent such as MeOH for other than silica, hexane for silica and remove air under pressure lower than half of what used in usual analysis. After the entire air is removed, check the performance by tracing the conditions on the inspection report which is attached with the product at the time of purchase.
- Inappropriate mobile phase conditions
- It may become difficult to obtain reproducibility in ionic compounds analysis if pH of mobile phase is not controlled or buffer concentration is low. Increase the buffer concentration.
- Retention time fluctuates widely due to a slight variance of pH in cases where the pH of mobile phase is set close to the pKa of analyte. Set the pH of mobile phase distant from pKa.
- System variance
- It may become difficult to obtain reproducibility in chromatogram by using different systems. Manufacturer of pumps, detectors and injectors should be the same, or extra column volume such as mixing chamber, detector cell and plumbing will be different and fail to obtain reproducibility between systems. Also, with column heater from different manufacturer may affect the retention time due to the required temperature difference between systems. Using a same system through out a sequence of analysis is recommendable.
- Column histories
- Reproducibility of chromatogram may not be obtained between the same type of columns. This is due to the column histories. For example, in some cases, change in surface condition of packing material that are caused by using columns with mobile phase containing ion pair reagent or adsorption of highly hydrophobic substances fails to obtain reproducibility. Dedicating a column per separation purpose is recommendable.
- Using 100% aqueous mobile phase
- Reproducibility of chromatogram on ordinary ODS columns will not be obtained by using 100% aqueous mobile phase due to a short retention time. Columns, can be used in 100% aqueous mobile phase, are recommendable and available from every manufacturer. For YMC columns, “Triart C18”, “Hydrosphere C18” and “ODS-AQ” can be used in 100% aqueous mobile phase.
- Grade difference in mobile phase
- Reproducibility of chromatogram may not be obtained by using different grade of solvent in mobile phase. Impurities contained in solvent can act like salts in mobile phase and affect the separation. HPLC grade solvent is recommendable.
This is caused by excess of ion pair reagent. In general, the concentration of ion pair reagent is higher, the stronger retention is observed. But in cases where the concentration of ion pair reagent is above a certain level, the retention may become poor because of micell formation. Good separation is achieved with the concentration of ion pair reagent, 5 mM to 20 mM. Set the concentration as low as possible to avoid short column life due to high ion pair reagent concentration.
Feature of columns / materials
We have 15 different kinds of C18 columns. They are divided in 4 groups, Hybrid silica based YMC-Triart columns, Core-shell type silica based Meteoric Core columns, Silica-based columns and Polymer-based YMC-Pack PolymerC18.
YMC-Triart columns exhibit extraordinary stability, low operating pressure, and excellent performance. Meteoric Core columns provide outstanding resolution compared to fully porous silica based columns.
To break down further within the Silica-based columns, they are divided in 3 groups, Pro series, YMC-Pack series (excl. PolymerC18) and J’sphere series. Pro series features in a superior performance and excellent reproducibility. Pro series with an efficient endcapping technology is superior to YMC-Pack ODS series and J’sphere series. You can find more information in the following C18 column selection guide.
ODS-A and AM are conventional ODS. ODS-AQ provides a lower rate of carbon content and is suitable for separation of hydrophilic compounds. ODS-A and -AM have the same basic physical properties such as the base material, the rate of carbon content and the separation characteristics, however -AM is produced under more strict Standards of Quality Control.
Polyamine II and PA-G are chemically bonded with polyamine where NH2 is with aminopropyl group. Polyamine II and PA-G are superior to NH2 in durability. They also have difference in selectivity. Polyamine II and PA-G have different ligand structure of polyamine. Polyamine II is superior to PA-G in durability.
General ODS (C18) packing material is a silica gel bonded with octadecyl groups. This is in the result of a reaction between silanol groups and octadecyl groups on the silica surface. However some silanol groups remain after the reaction. It is impossible for all the silanol groups to react because of steric hindrance of octadecyl groups. Such residual silanol groups create a secondary interaction in chromatography, which, in many cases, affects on chromatograms by, in general, causing a peak tailing of basic compounds or irreversible absorption to the column. Therefore, a secondary silanization on residual silanol groups with unbulky silanization reagents should be performed. This process is called “endcapping”. Trimethylsilane (TMS) is commonly used in “endcapping” process.
“Triart C18”, “Hydrosphere C18” and “ODS-AQ” columns can be used with 100% aqueous mobile phase. On conventional ODS columns, retention time is shortened due to the incompatibility between water and material surface with high hydrophobicity. Water tends to be expelled from the pores on material. The retention time hardly shortened on “YMC-Triart C18”, “Hydrosphere C18” and “ODS-AQ” because they are capable of solvation between mobile phase and hydrophilic surface by reducing the density of C18 functional groups.
Column handling
Applicable pH range and temperature
Column Type | pH range | Usable temperature range | |
---|---|---|---|
Regular use | Upper limit | ||
Triart C18, C18 ExRS, C8 | 1 – 12 | 20-40℃ | pH 1-7 : 90℃ pH 7-12 : 50℃ |
Triart Phenyl | 1 – 10 | 20-40℃ | 50℃ |
Triart PFP | 1 – 8 | 20-40℃ | 50℃ |
Triart Bio C18 | 1 – 12 | 20-40℃ | pH 1-9 : 90℃ pH 9-12 : 50℃ |
Triart Bio C4 | 1 – 10 | 20-40℃ | pH 1-7 : 90℃ pH 7-10 : 50℃ |
Pro C18, Hydrosphere C18 | 2 – 8 | 20-40℃ | 50℃ |
Pro C18 RS | 1 – 10 | 20-40℃ | 50℃ |
J’sphere ODS-H80 | 1 – 9 | 20-40℃ | 50℃ |
PolymerC18 | 2 – 13 | 25-35℃ | 65℃ |
Triart Diol-HILIC | 2 – 10 | 20-40℃ | 50℃ |
Reversed-phase (Other than mentioned above) Normal phase (SIL, Polyamine II) | 2 – 7 | 20-40℃ | 50℃ |
*The given data is subject to change depends on product types. Those data should be confirmed with Instruction manual when the column is used.
When columns are not used for a long time, keep them in a cool place after replacing with the shipping solvent as indicated in the attached inspection report. Do not keep the column in the mobile phase with salt or acid regardless of whether or not it is in a short period of time. Close the airtight stopper tightly to prevent the solvent from volatilizing.
Perform an inspection test under the same conditions as the inspection report attached to the column at the time of purchase. Columns are evaluated to be effective and have no change in performance if the result indicates no irregularity in retention time, theoretical plate number, peak asymmetry, etc. Columns which indicate no irregularity in the said criteria after using several years from purchase, however, may have changes in separation characteristics for compounds such as ionic compounds.
It is advisable to avoid using them for method development. Reproducibility may not be obtained with new columns.
Triart Series: Acetonitrile (100)
Pro Series, ODS-A, AM, AQ, etc.: Acetonitrile/Water (60/40)
J’sphere Series: Acetonitrile (100)
* Indicated in the COLUMN INSPECTION REPORT.
- Remove highly hydrophobic substances adsorbed onto the gel
- Use solvent with a stronger elution ability than mobile phase. For example, cleaning reversed-phase columns, use solvent with increased ratio of organic modifier and flush the volume of 10 times as much as the column volume.
- Renaturation of gel surface condition
- Irregularities are observed in Peak asymmetry and retention time.
In silica-based packing material, separation behavior may be affected by the conditions of residual silanol groups whether in dissociation or in non-dissociation. Washing with acidic solvents may be effective in such case. Washing with a mixed solvent of 0.1% aqueous phosphoric acid solution and organic solvent* can perform the renaturation of silanol groups to the dissociation state.
* Ratio of organic solvent: 10 to 60%.
- Irregularities are observed in Peak asymmetry and retention time.
To analyze samples contain a lot of contaminants, guard column is effective and can improve the durability of main columns. We recommend guard columns with the same packing materials as main columns. Guard columns with different material may cause defects in peak asymmetries and reproducibility. We have them in 2 types, conventional type and cartridge type. We recommend cartridge type if guard columns require a frequent replacement. Inner diameter should be same as the main column or smaller.
“WT” indicates Waters connector compatible, “PT”, “PTH”, “PTC” or “PTP” indicates Parker connector compatible. The majority of columns in the market are of these types. There are several connection types other than Waters and Parker compatible types such as Shimadzu, JASCO, Hitachi, etc. The difference in these connection types is the length of tubing section coming out from the tip of ferrule. The connector types of column and tubing system should be the same, or tubing and column may fail to fit well and cause leakage and defects in peak asymmetry. If your system has something other than Waters, a connection adapter or a ferrule replacement may be required. * PEEK inch screw thread built-in ferrule would not have the problem.
The end of the product number | *Port depth | Style of endfitting |
PT/ PTH/ PTC/PTP | 2 mm | Parker style (UPLC compatible) |
WT/ WX/ WTG/WP/WTS | 3 mm | Waters (W) style |
UPLC is a registerd trademark of Waters Corporation
Flow rate on Semi-micro column (hereafter columns in 1.0 to 2.0 mm inner diameters will be mentioned as semi-micro columns) is 50 to 200 µL/min in general. It can be increased if the length of column is short and back pressure is low. Commonly used HPLC System is applicable, however, with pumps, flow cell of detectors and tubing system designed for semi-micro column is more suitable.
- Step 1
- Determine separation conditions by using analytical columns.
- Step 2
- Study the preparative scale. Set the particle size of the packing material and the inner diameter of column in consideration of the sample volume.
- Step 3
- Optimize the separation conditions by using analytical columns with inner diameter of 4.6 mm or 6.0 mm packed with the selected packing material. If the particle size of the packing material is the same as in the Step 1, this process can be omitted. If the preparative scale is more than 100 mm in inner diameter, another process using a column in a diameter of 20 mm will follow to determine the loadability and calculate the running cost.
- Step 4
- Proceed with the preparative separation.
Solution of column trouble
Wash the column under the method in “How to clean the columns? 1. Remove highly hydrophobic substances adsorbed onto the gel” Reduce the flow rate accordingly in order to keep the column pressure adequate when flashing the column. If the cause is believed to clog frit or terrible contamination, washing by reversed direction flow will be very effective.
* If pressure increase is observed often after washing the column, take such measures as sample pretreatment or using guard columns to prevent the problem
Trouble Shooting tips : If Column Pressure Increases
- Inappropriate Mobile phase
- In the case of ionic analyte if pKa of the analyte and pH of mobile phase are close, it causes defects in the peak shape.
Set the pH of mobile phase distant from pKa.
- In the case of ionic analyte if pKa of the analyte and pH of mobile phase are close, it causes defects in the peak shape.
- Influence by dissolving solvent of sample
- If dissolving solvent of sample and mobile phase are not the same, it causes defects in the peak shape. Dilute the sample solution with mobile phase or reduce the injection volume.
- Overloading sample injection
- Overload will cause defects in the peak shape.
Reduce injection volume.
- Overload will cause defects in the peak shape.
- Insufficient equilibration time
- When variance in pH is wide between the current and previous mobile phase or the buffer concentration of mobile phase is low, column equilibration may take a while.
- Column contamination and degradation
- In the case of contamination, wash the column according to “How to clean the columns? 1. Remove highly hydrophobic substances adsorbed onto the gel”. If column is in degradation, it is impossible to regenerate. The column should be replaced.
- System problem
- Dispersion may occur within tubing between injector and column or the flow cell of detector which results in peak tailing and/or broading. System should comply with semi-micro use.
Trouble Shooting tips : Peak shape anomaly
Following solutions are introduced depends on causes.
- Injector fouling (carry-over)
- If the ghost peak appears when injecting mobile phase only, wash the injector.
- Gradient Analysis
- When hydrophobic impurities are eluted by a stronger solvent, it appears as a ghost peak. Clean the column according to “How to clean the columns? 1. Remove highly hydrophobic substances adsorbed onto the gel”. If you still can’t eliminate them, the cause should be impurities of solvent.
Use a higher grade solvent. Trap the impurities by attaching guard column between the solvent delivery pump and the mixing chamber.
- When hydrophobic impurities are eluted by a stronger solvent, it appears as a ghost peak. Clean the column according to “How to clean the columns? 1. Remove highly hydrophobic substances adsorbed onto the gel”. If you still can’t eliminate them, the cause should be impurities of solvent.
Trouble Shooting tips : The Cause of the Ghost Peak
Flush the column with solvent such as MeOH for other than silica, hexane for silica and remove air under pressure lower than half of what used in usual analysis. After the entire air is removed, check the performance by tracing the conditions on the inspection report which is attached with the product at the time of purchase.
- Inappropriate mobile phase conditions
- It may become difficult to obtain reproducibility in ionic compounds analysis if pH of mobile phase is not controlled or buffer concentration is low. Increase the buffer concentration.
- Retention time fluctuates widely due to a slight variance of pH in cases where the pH of mobile phase is set close to the pKa of analyte. Set the pH of mobile phase distant from pKa.
- System variance
- It may become difficult to obtain reproducibility in chromatogram by using different systems. Manufacturer of pumps, detectors and injectors should be the same, or extra column volume such as mixing chamber, detector cell and plumbing will be different and fail to obtain reproducibility between systems. Also, with column heater from different manufacturer may affect the retention time due to the required temperature difference between systems. Using a same system through out a sequence of analysis is recommendable.
- Column histories
- Reproducibility of chromatogram may not be obtained between the same type of columns. This is due to the column histories. For example, in some cases, change in surface condition of packing material that are caused by using columns with mobile phase containing ion pair reagent or adsorption of highly hydrophobic substances fails to obtain reproducibility. Dedicating a column per separation purpose is recommendable.
- Using 100% aqueous mobile phase
- Reproducibility of chromatogram on ordinary ODS columns will not be obtained by using 100% aqueous mobile phase due to a short retention time. Columns, can be used in 100% aqueous mobile phase, are recommendable and available from every manufacturer. For YMC columns, “Triart C18”, “Hydrosphere C18” and “ODS-AQ” can be used in 100% aqueous mobile phase.
- Grade difference in mobile phase
- Reproducibility of chromatogram may not be obtained by using different grade of solvent in mobile phase. Impurities contained in solvent can act like salts in mobile phase and affect the separation. HPLC grade solvent is recommendable.
This is caused by excess of ion pair reagent. In general, the concentration of ion pair reagent is higher, the stronger retention is observed. But in cases where the concentration of ion pair reagent is above a certain level, the retention may become poor because of micell formation. Good separation is achieved with the concentration of ion pair reagent, 5 mM to 20 mM. Set the concentration as low as possible to avoid short column life due to high ion pair reagent concentration.
Troubleshooting Tips
Reversed-phase liquid chromatography frequently employs such organic solvents as methanol, acetonitrile or tetrahydrofuran. Although HPLC grade products of these types of solvents are available, it seems some users have trouble when using a reagent grade solvent instead of HPLC grade, leading to them wasting considerable amounts of time. How do the two solvent grades differ?
Methanol/acetonitrile
Reagent grade solvents contain larger quantity of impurities absorbing UV than HPLC grade solvents do, which makes it difficult to use them in gradient elution or trace analysis. Especially when detection is conducted in a short wavelength, significant differences appear in baseline noise or detection sensitivity. In some cases (or in some wavelengths) it could be feasible to use a reagent grade solvent but we recommend HPLC grade solvents to obtain a stable chromatogram.
Tetrahydrofuran
Tetrahydrofuran easily generates peroxides. To compensate for this tendency, it is commonly mixed with antioxidants. The antioxidants cause a ghost peak so a solvent not containing antioxidants should be used in HPLC. The peroxides in tetrahydrofuran also have great impact on the baseline stability (with differences between grades greater than those of other organic solvents), which prompts a strong recommendation to use HPLC grade solvents with very small quantities of impurities.
Although UV detectors are most commonly used in HPLC, refractive detectors are used when analyzing a compounds without a UV absorption band such as sugars. When conducting sugar analysis using a refractive detector, baseline instability will generally become a problem. Probably most of the baseline drift problems are due to the changes in temperature of a column. Comparison of baseline stability by a difference of a control method of column temperature is shown in the figures below. Baseline drift is very high at an ambient temperature without any column temperature control. Even in the case of a water bath or a column oven, baseline noise may occur by the change of temperature as a result of ON/OFF of a heater. As a measure to avoid this, placing the column in a water bath at the ambient temperature and stirring without heating can produce the desired effect.
Analysis of ionic compounds by reversed-phase HPLC requires the pH control of eluent with acid or buffering agent. The pH control stabilizes the dissociation state of compounds and enhances the reproducibility of retention and separation. Unsuitable pH in analysis causes problems such as a double peak or peak broadening. As shown in the figure below, an identical compound is eluted showing the double peak in pH4.8 eluent, while showing a sharp peak in pH7.0. When analyzing an ionic compound, finding the right pH range for each functional group will result in avoiding such problems.
HPLC analysis of bio samples requires complicated sample preparation depending on the component of interest and sometimes calls for very small amounts of sample for injection. If the injection mass is extremely small in a column in general use with internal diameter of 4.6mm, the component of interest might not be detected as a peak due to diffusion of substance in the column. A semi-micro column with a small internal diameter will be helpful in detecting substances in such small amounts. It is also helpful for eluent saving or application to LC/MS because lower flow rates can be selected in analytical operations.
Although the semi-micro column is helpful as mentioned above, care must be taken during operations as described below. Factors causing poor column performance include dead volumes of tubing system including flow cells of the detector.
The figures below depict how internal diameters of tubes and internal volumes of flow cells affect chromatograms. Given the tubing system compatible to analytical columns with standard sizes, column performance (theoretical plate or peak symmetry) degradation occurs due to sample diffusion outside the column. Using semi-micro columns requires suppression of sample diffusion by the tubing system. On the other hand, it should be noted that a small flow cell volume forces a path length to shorten resulting in a lower peak height. It is important to arrange the system environment keeping in mind the considerations discussed above to obtain the column performance and detection sensitivity appropriate to separation of interest.
Are you overly concerned about the degree of absorption when you set the detection wavelength verifying the UV absorption of the component of interest?
Some substances greatly alter their absorption coefficients with a 2 to 3nm variation in the detection wavelength. Moreover, there are instrumental errors. In other words, however precisely you set the wavelength there must be small deviations. To avoid making the 2 to 3 nm deviation matter too much, it is important to choose a stable wavelength with little deviation of the absorption coefficient near the set wavelength rather than choose an unstable wavelength with high absorption.
YMC-Pack Polymine II is highly effective in separation of saccharides including oligosaccharides and is employed in normal phase separation with nonaqueous eluent or in separation of ionic compounds by combining normal phase and weak anion exchange mode.
In the example of analysis of ascorbic acid depicted below, anions in the eluent and amino groups on the surfaces of support material are in a state in which ion-pairs are formed. If the ion-pairing has not yet reached ionic equilibrium, retention times of a sample might alter. We recommend that to avoid this problem by facilitating ion-pairing, the operation as described below be conducted before starting analysis.
Column equilibration in ascorbic acid analysis (in the case the column size 250 X 4.6mm I.D.)
- Flush the columin with water at the flow rate of 1.0 mL/min for 10 minutes.
- Flush the column with 200mM of water solution of ammonium dihydrogen phosphate at the flow rate of 1.0mL/min for 40 minutes.
- Flush the column with water at the flow rate of 1.0mL/min for 30 minutes.
- Flush the column with the eluent for analytical use at the flow rate of 1.0mL/min for 60 minutes.
After using these eluents, sugar analysis using acetonitrile/water eluents might cause anomalies in peak shapes (such as doubling or broadening). Considering the life time or separation reproducibility, it is desirable that separate columns be respectively used for each eluent.
Common problems during HPLC operations include peak shape anomalies such as peak tailing and double peaks. To address these problems, the cause must be precisely determined. The majority of cases are caused by inappropriate conditions of separation which include improper selection of a column or solvent, or deficient columns such as those with void. Here we introduce the method of determining which is the cause.
The simplest way is to examine the column performance according to “shipping inspection criteria” in the column inspection report attached to the column. If the examination reveals no peak shape anomalies, the cause will be an inappropriate separation condition. The separation condition such as eluent selection must be reconsidered.
If on the