Instruction manual for Chiral CD-Ph

• 1. Column Handling
• 2. Column Installation
• 3. Analysis
• 4. Storage of columns
• 5. About the column connection
• 6. Troubleshooting

The Chiral CD-Ph is a HPLC column designed with phenylcarbamated β-cyclodextrin bounding to the precisely-graded high-purity spherical silica substrate by a spacer moiety.

1. Column Handling

1. Handle the column with great care. Strong shock may cause damage.
2. Attach or detach the column when the pressure gage indicates zero.

2. Column Installation

1. The column joint is a male-nut type for 1/16 inch OD tubing. In the case of SUS specifications, make sure that the tubing joints of the system fit correctly and that the ferrule tips are inserted completely into the joints. (See Fig. 1.)
2. Before attaching the column, displace the liquid in the system with the mobile phase to be used.
3. Before attaching the column, displace the liquid in the system with the mobile phase to be used.

3. Analysis

3-1. Mobile phase

1. All types of solvents usable for chemically bonded silica can be used.
2. The range of acceptable pH of Chiral CD-Ph columns is from 3.5 to 6.5. In order to prevent premature deterioration of columns, please ensure that the mobile phase pH will not exceed this range.
   Unlike CAPCELL PAK, Chiral CD-Ph is not compatible with basic mobile phases.
3. After ensuring the absense of salt precipitation and fully degassing, remove insoluble matter and foreign particles by filtering through a membrane filter (0.45 µm or less). Incidentally, with a view to preventing clogging of the column inlet filter by foreign matter, it is recommended that a liquid line filter be used.
4. A testing mobile phase is enclosed with the column for shipping. Displace the shipment mobile phase sufficiently with ethanol or isopropanol before use with a hexane-based mobile phase.
5. Avoid the following practices or factors because they generally lead to the deterioration of columns.
   • Frequent changes of the composition of mobile phase or a direct change to a mobile phase of poor compatibility
   • Sudden change of pressure at the column inlet
   • High column pressure due to use of a high-viscosity mobile phase
6. The maximum pressure for regular use of Chiral CD-Ph is 20 MPa. When using a mobile phase of high viscosity or when measuring at a low temperature, pay utmost attention to the pressure.
7. In order to achieve the stable retention time with Chiral CD-Ph, a step of at least 3 hours of equilibration is required.

3-2. Preparation of sample solution

1. Preferably, dissolve the sample in a solvent of the same composition as that of the mobile phase.
2. Be aware that use of a strong solvent may lower resolution or cause the sample to precipitate at the column head.
3. If insoluble matter remains in the sample solution, filter through a filter (0.45 µm or less)

3-3 Procedures for optical separation

1. Temperature
   It is recommended that work be started at room temperature (25 °C) . Incidentally, the packing material is stabilized at below 80 °C.
2. Flow rate
   It is recommended that flow rate be started at 500 µL/min
3. Mobile phase
   1. Nonionic compounds
   It is recommended that optical separation procede in the following order: isopropanol/hexane, ethanol/hexane, methanol/water, ethanol/water, acetonitrile/water. Adjust the compounding ratio of polar organic solvents (methanol, ethanol, isopropanol, acetonitrile) by decreasing it gradually from 90 % so that the retention time of target compound falls within the range of 7 to 30 min.
   2. Ionic compounds
   We recommend an aqueous solution of 20-50 mmol/L potassium dihydrogenphosphate and a polar organic solvent. Sodium perchlorate and triethylamine acetate are also effective substitutes for potassium dihydrogenphosphate. It is recommend that optical separation proceed in the order of acetnitrile and then ethanol.

3-4 Points to note in the analysis of basic compounds

The action mode for retention and separation of basic compounds in optical separation is different from the mode in the common ODS columns of a reversed phase system. Consequently, due to its characteristic features, there are certain cases in which variation among product lots may not be easily identified only by the specifications provided. Therefore, when optical separation is applied to basic compounds, it is recommended that samples from a number of lots be evaluated.

4. Storage of columns

1. Hermetically seal with accessory plugs and store in a cold place where temperature fluctuation is negligible.
2. If the column is to be stored for an extended period subsequent to the use of a mobile phase containing an inorganic salt like sodium perchlorate, displace with an aqueous solution of methanol of 70 v/v %.

5. About end-fitting

1. Chiral CD-Ph uses an end-fitting with the structure shown in Fig. 1. Replacement of filter alone is not possible.
2. For tubing, follow the instructions in Fig. 1. Inappropriate tubing, particularly when a tube used for a different type of column is used as is, the tip length beyond the ferrule (V in Fig. 1) may not match the length of the end fitting bore L , which leads to troubles.
   Where L>V, dead volume occurs and may cause the broadening or tailing of peak, or limit separation performance.
   Where L<V, inadequate sealing of the ferrule causes liquid leakage. Therefore, it is recommended that the ferrule be changed concurrently with the column change.
   * After frequent column replacement the male-nut may present a crushed ferrule and cause liquid leakage. In such a case, tightening too hard may cause the head portion of the nut to break off. Therefore, replace the ferrule in good time.



Fig. 1

6. Troubleshooting

Since it is impossible to enumerate all of the troubles associated with HPLC analysis work have diverse causes, only those which occur relatively often in and around the column are cited:

Symptom	Causes	Remedies
1. Rise in column pressure	Clogging with foreign matter 1. Foreign particles, insolubles in mobile phase and sample solutions 2. Water deposits in the tubing 3. Fragments of plunger seal 2. 4. Precipitation of sample constituents	• Filter the mobile phase and sample solution through a membrane filter beforehand. • Filter the mobile phase and sample solution through a membrane filter beforehand. • Install a liqid line filter. • Clean the tubing and replace the plunger seal • Prepare the sample solution with the mobile phase
2. Splitting, tailing or broadening of peak	1. Occurrence of void (cavity) in column head 2. Occurrence of dead volume due to faulty tubing 3. Inappropriate conditions of mobile phase 4. Deterioration of column * Restoration is impossible when deterioration of column or void in packing material bed has occurred.	• Dismantle the tubing once and reconnect. • Ascertain pH, salt concentration, sample volume, etc. • Verify column performance using the standard testing solution.
3. Retention time too long or unstable	1. Liquid leakage (this can be judged by the movement of pressure gauge on the pump) 2. Inappropriate conditions of the mobile phase 3. Insufficient time for column stabilization	• Inspect the liquid leakage from pump and tubing system. See 2-3 • Take sufficient time for stabilization.
4. Retention time too short	1. Rupture (deterioration) of bonding group due to the use of strong acid or alkali 2. Inappropriate conditions of mobile phase 3. Insufficient time for column stabilization	See 2-3 • Take sufficient time for stabilization.

While Chiral CD-Ph undergo strict verification of performance before shipping, if any defect is identified, please inform us or our distributor of it. We will replace it.
However, please take note that we are not responsible for matters related to column service life or problems caused by the failure to follow the above mentioned instructions for care and use. Please note also that we assume the product is assumed to have been delivered in good condition if no claim is made within ten days from receipt by the customer, and requests for replacement submitted thereafter will not be accepted.

Revised June 21st 2011