This is the Part 7 of our following series “Q&A about Liquid Chromatography” that was published before. Which will cover some more Q&A, help you to know more about liquid Chromatography.

Q1:Influence of column packing pore size on sample detection
A: The pore size of the column is determined by the molecular weight. The influence of the pore size of the packing on the resolution. The 120 Å chromatographic column is usually applicable to the column with molecular weight less than 2000. If the molecular weight is too large, the resolution will be poor on the column with the pore size of 120 Å, because the sample molecules are well retained on the chromatographic column because they can enter the micropores of the packing and interact with the C18 long chain bonded on the surface, Usually, the pore diameter needs to be more than 3 times larger than the molecular diameter in order not to affect the analysis. Therefore, the molecular weight of general chemical drugs is not more than 2000, biological drugs are not more than 5000, 120 Å is used, chemical drugs are more than 2000, and biological drugs are more than 5000, 300 Å is used.

Q2: General method for maintenance of chromatographic column after trial use of different buffer salts


When buffer salts are contained in the mobile phase, the general principle is: transition before use, cleaning after use (cleaning includes two steps, one step is used to clean buffer salts, the other step is used to clean strong retention substances), please maintain the column as follows.

Transition before use:

Acetonitrile: water = 10:90 (methanol: water = 10:90 if methanol system) was used for 30min transition at a flow rate of 1mL/min, about 7 column volumes (the purpose is to prevent buffer salt precipitation when entering the column).

After one day analysis, immediately after use cleaning:

Step 1) Acetonitrile: water = 10:90 (or methanol: water = 10:90) at the flow rate of 1mL/min, reverse rinse for 45min, about 10 column volume (the purpose is to wash buffer salt, prevent buffer salt in the column to reduce the service life);

Step 2) Then use acetonitrile: water = 80:10 (or methanol: water = 80: 10) 1 ml/min flow reverse flushing 45 min, approximately 10 column volumes (purpose is to wash the analysis in the process of accumulation of retained substances, prevent the strong reserve substance accumulation within the chromatographic column, the analysis of the influence after also prevent strong retention caused by the accumulation of material column pressure), and then save the chromatographic column in acetonitrile: water = 80:10 (or methanol: Water = 80:10) in.


1) Acetonitrile: water = 10:90 or methanol: water = 10:90 is easy to grow bacteria, the higher the temperature is, the more easy to grow bacteria, the experiment shows that when stored at 28 degrees to the fifth day, the growth of bacteria is found, it is not suitable to mix more.

2) Welch’s most chromatographic columns can be backwashed or used (the column specification will specify which column can recoil, if not, can not rebound). If the column you use can not be backwashed or used, there is no recoil. This method is also applicable, but the rebound effect is relatively good.

Q3: The difference between the use of normal phase and reverse phase of amino column and the preservation of chromatographic column:


Positive phase system:

It is recommended to first use n-hexane-isopropanol (99: 1) with a flow rate of 0.5 ml / min, and then use the flow phase similar to the path according to the flow, the same flow rate rushed 10 times the volume, and finally replaced Mobile phase. When used in use, it is not appropriate to analyze the compound containing aldehyde group, a carbonyl group, and is not used for the analysis of reducing sugar; the flow phase should be thoroughly degassed, and the carbonyl compound and peroxide (diethyl ether, tetrahydrofuran containing a small amount) .

Inverted system:

First use isopropyl alcohol by a flow rate of 0.5 ml / min, the same amount of acetonitrile in the same amount of flow rate, in turn, in the same amount of flow rate, in the transition to the flow phase system. Replace it into a mobile phase. When used under reverse phase conditions, pay special attention to control the pH range, the lower the pH, the more hydrolyzes, the higher the proportion of water in the flow phase, and the more dangerous hazards are also there. The most desirable pH range is in the range of pH 2.5 to 8, which is recommended to use the water phase <40% or less.

Reverse use save:

Under positive phase, the flow phase balance system can be used directly, and after use with isopropanol, it is flushed with 0.5 ml / min flow velocity, and finally stored in isopropyl alcohol and is used in forward use.

Reverse use save:

Used under reverse phase conditions, first use isopropyl alcohol with a 0.5 ml / min flow rate of 60 min, then rinse with methanol or acetonitrile 30min for 30 min to transition to inverting mode. Then replace the flow phase to perform a balance if there is a buffer salt in the flow phase, and the organic phase and the aqueous phase of the same equivalent ratio using the same flow is used before use, but the aqueous phase does not contain buffered salts. After 30 minutes of the transition, change it to the mobile phase for balance column.

Q4: Operation flow of lactose detection using amino column with large baseline fluctuation


1) Activation of new column: Wash with Isopropanol at the flow rate of 0.5ml/min for 4 hours, then pure acetonitrile and 70% acetonitrile water at the flow rate of 1ml / min for 1 hour respectively, and then wash with mobile phase to the baseline for stable sample injection.
2) Mobile phase configuration: It is recommended to premix the two mobile phases. Before mixing the mobile phases, filter the aqueous phase and organic phase with water membrane and organic phase membrane respectively, and then prepare and mix 500ml mobile phase in the same solvent bottle according to the proportion. The mobile phase is in the same solvent bottle (premixed manually). Shake the solvent bottle fully to make the two phases mix evenly. Degassing with ultrasonic for 20 minutes, If there are signs of heating, place back to room temperature.
Put the mobile phase on the instrument, turn on the power supply of the instrument pump, detector and on-line degasser, set the column temperature: 45 ℃, detector temperature: 40 ℃, flow rate: 1ml / min, turn on the reference tank of rid detector, after flushing for 30min, turn on the circulation button of differential detector to make the outflow liquid flow out of the recycle pipeline (after the liquid in the return pipe flows out for 20min), Then place the return pipe in the mobile phase bottle for mobile phase circulation.
3) After one night of circulation, switch the mobile phase circulation to the waste flow path the next day, close the reference cell, flush the sample cell for 30min, the baseline is stable and the sample injection is tested.
4) If lactose at the concentration of 1mg / ml still does not peak, configure a concentration of 5mg / ml.
Note: When circulating at night, it must be the setting of mobile phase chromatographic conditions of analytical samples

Q5: Precautions and maintenance of Welch Xtimate ®sugar Ca and Sugar-H chromatographic columns.


Xtimate® SUGAR-CA, SUGAR-H, packing belongs to polystyrene – diethylenylbenzene polymer matrix, sulfonated strong cation exchange resin, the first thing to avoid during the use of mobile phase reagent contamination of the column, especially to avoid the use of pure organic solvent washing caused by swelling column packing. The separation effect of chromatographic column was lost due to the change of packing properties.

Sugar-Ca can be used to determine mannitol and sorbitol in pharmacopoeia, as well as carbohydrates, monosaccharides or polysugars. The mobile phase system and sample solvent are basically pure water system.

Sugar-H is designed for the determination of ribavirin in pharmacopoeia and can also be used for the determination of carbohydrate or organic acid separation using sulfuric acid aqueous solution with a pH of around 2.5 as the mobile phase. After use, the mobile phase is basically sealed at a low temperature of 4 degrees. In the process of sealing, there will be strong bacteria if the time is too long. Therefore, it is better to use a new mobile phase for sealing for a period of time (2-3) weeks.

Sugar-Ca column regeneration steps:

When the shape of the main peak changes and the column efficiency decreases, regeneration is needed.

The sugar-CA guard column regeneration method is consistent with the SUGAR-CA analysis column method. The more natural the sample (especially those containing heavy metals and transition metals or organic acids), the more frequently the column will need to be regenerated because they will displace the column. Column displacement can be tested by injecting sucrose. If the peak of sucrose is trailing, the column should be treated as follows: prepare 500mL recycled solution (Ca EDTA 500mg/L Cas:23411-34-9), reverse the flow direction of the column and wash it overnight at 85℃ at a flow rate of 0.5mL/min. Return to normal direction after regeneration.

Sugar-H column regeneration steps:

The method of regenerating sugar-H columns varies depending on the purity of the sample concerned, and the more natural the material (especially those containing heavy metals and transition metals or organic acids), the more frequently the columns need to be regenerated as they will displace the column. If the main peak has a tail, the column needs to be treated as follows: prepare 500mL recycled solution (dilute sulfuric acid with pH2.5), reverse the flow direction of the guard column and analysis column as a whole, and wash overnight at 85℃ at a rate of 0.5mL/min. When the column recoil, it is important to maintain the temperature of the solvent to ensure proper washing. Using a cold solvent will delay the washing process.

If you have any problem or require further information, please contact info@welchmat

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