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Feature of columns/materials

What are the features of YMC C18 Columns?

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.

How different are ODS-A, AM and AQ?

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.

How different are YMC-Pack Polyamine II, PA-G and NH2?

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.

What is "Endcapping"?

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.

Are there C18 columns used with 100% aqueous mobile phase?

"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

What is the upper limit of column pressure?

Recomended upper limits of column pressure is as follows.

General (other than specified below)
Column length of 150 mm or less : Approx. 20 MPa
Column length of 250 mm : Approx. 25 MPa
Inner diameter of 10 mm or more : Approx. 10 MPa

  • YMC-Triart 1.9 µm : Approx. 100 MPa
  • YMC-Triart 3, 5 µm (PTH type) : Approx. 45 MPa
  • Meteoric Core : Approx. 60 MPa
  • UltraHT Series Column length of 50 mm or more : Approx. 50 MPa
  • UltraHT Series Column length of 30 mm : Approx. 40 MPa
  • Actus Series : Approx. 30 MPa

*Indicated in the instruction manual.

What are the applicable pH range and temperature?

Applicable pH range and temperature

Column Type pH range Usable temperature range
Regular use Upper limit
Triart C18, C18 ExRS, C8 1.0 - 12.0 20-40℃ pH 1-7 : 70℃
pH 7-12 : 50℃
Triart Phenyl 1.0 - 10.0 20-40℃ 50℃
Triart PFP 1.0 - 8.0 20-40℃ 50℃
Pro C18, Hydrosphere C18 2.0 - 8.0 20-40℃ 50℃
Pro C18 RS 1.0 - 10.0 20-40℃ 50℃
J'sphere ODS-H80 1.0 - 9.0 20-40℃ 50℃
PolymerC18 2.0 - 13.0 25-35℃ 65℃
Triart Diol-HILIC 2.0 - 10.0 20-40℃ 50℃
Reversed-phase (Other than mentioned above) Normal phase (SIL, Polyamine II) 2.0 - 7.5 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.

How should we store the columns?

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.

How can we evaluate the performance of columns?

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.

What is the shipping solvent?

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.

How to clean the columns?

  1. 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.
  2. 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%.

Do we need Guard columns?

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.

What is the last letters of product code "WT", "PT", "PTH" and "PTP"?

"WT" indicates Waters connector compatible, "PT", "PTH" and "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.

What is required in system and flow rate for using semi-micro columns?

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.

How to scale-up a column for preparative isolation/purification?

Carry out a scale-up in the following procedure.

  • 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

What should I do when the column pressure rises up?

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

What are the solutions for poor peak shapes?

Following solutions are introduced depends on causes.

  1. 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.
  2. 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.
  3. Overloading sample injection
    Overload will cause defects in the peak shape.
    Reduce injection volume.
  4. 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.
  5. 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.
  6. 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

What are the solutions for ghost peaks?

Following solutions are introduced depends on causes.

  1. Injector fouling (carry-over)
    If the ghost peak appears when injecting mobile phase only, wash the injector.
  2. 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.

Trouble Shooting tips : The Cause of the Ghost Peak

What should I do if columns are dried?

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.

What should I do if the column fails to provide reproducibility?

  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. 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.

I still have poor retention after adding ion pair reagent to mobile phase. Why?

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.