我們總共有超過15種不同材質的C18管柱，可分為四種類型。使用混合層(Hybrid)矽膠之YMC-Triart系列層析管柱, 實心核矽膠Meteoric Core 層析管柱, 矽膠材質(Silica-based)層析管柱以及聚合物材質(Polymer-based)PolymerC18層析管柱。 YMC-Triart系列層析管柱具有優越之穩定性、操作壓力低以及傑出的分離能力的特點。Meteoric Core 層析管柱與多孔性矽膠相比，則有出色的解析度特點。 矽膠材質(Silica-based)層析管柱可進一步細分為3群：Pro series, YMC-Pack series (除 PolymerC18)以及J'sphere series. Pro series 特色是優越的性能以及傑出的再現性。更多說明可參考C18 column selection guide.

ODS-A和AM是常見的ODS(Octadecyl-silica, C18)。 ODS-AQ的碳含量較低，適用於親水性化合物的分離。ODS-A和-AM具有相同的基本物理特性(例如基材、碳含量的比率和分離特性)，不過-AM是在更嚴格的品質控制標準下生產的。 ￭ YMC-Pack ODS-A ￭ YMC-Pack ODS-AM ￭ YMC-Pack ODS-AQ

Polyamine II 和 PA-G 使用polyamine 官能基之化學鍵結，NH2使用aminopropyl group化學鍵結。Polyamine II 和 PA-G 耐用性較NH2 佳。彼此的選擇性也不同，Polyamine II 和 PA-G 有不同的配位基(ligand)結構， Polyamine II 耐用性較PA-G佳. ￭ YMC-Pack NH2 ￭ YMC-Pack PA-G ￭ YMC-Pack Polyamine II

一般 ODS (C18) 膠體是指矽膠上有octadecyl(C18)官能基鍵結。這代表的是二氧化矽(silica)表面上的矽烷醇基(silanol groups)與octadecyl groups(C18)的鍵結。然而，反應過後仍會有部分未鍵結之矽烷醇基(silanol groups)。由於octadecyl groups(C18)的立體結構障礙(steric hindrance)，是無法讓所有的矽烷醇基(silanol groups)與octadecyl groups(C18)反應。未鍵結之矽烷醇基(silanol groups)會造成層析(chromatography)過程中發生二次反應(secondary interaction)，例如：鹼性化合物(basic compounds)拖尾問題(peak tailing)、管柱不可逆吸附(irreversible absorption)。因此，使用無立體障礙之矽烷化試劑(unbulky silanization reagents)進行未鍵結之矽烷醇基(silanol groups)進行第二次矽烷化(secondary silanization)反應。這樣的過程稱為封端(Endcapping)。三甲基矽烷(Trimethylsilane, TMS)為常見之Endcapping技術。

"Triart C18", "Hydrosphere C18" and "ODS-AQ" 可使用100%全水相之移動相。在傳統的ODS層析管柱上，由於水和疏水性高的膠體表面之間的不相容性，會縮短滯留時間(retention time)。水會傾向於從材料上的孔隙中排出。 在“ YMC-Triart C18”，“ Hydrosphere C18”和“ ODS-AQ”上的滯留時間(retention time)幾乎不會縮短，因為它們能夠藉由降低C18官能基的密度而使移動相和親水性表面互溶。 ￭ YMC-Triart C18 ￭ Hydrosphere C18 ￭ YMC-Pack ODS-AQ

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.

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%.

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" 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.

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.

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.

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

Following solutions are introduced depends on causes.

• 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.
• 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.
• 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.

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.