41. What is an ideal criteria for peak symmetry?
If not specified in the monograph or test method such as assay, related substance or other test,
the symmetry factor or tailing factor applicable for the peak during quantitative evaluation is 0.8 to 1.8.
42. What is a signal-to-noise ratio?
The signal to noise ratio represents the capability of a method to detect or quantify the elute in a consistent manner; in other word, the signal-to-noise ratio represents the system sensitivity.
When the signal-to-noise ratio is more than 3, compounds can be consistently detected for each time. When the signal-to-noise ratio is more than 10, compounds can be consistently quantified for each time
43. What are the issues if the Reverse Phase HPLC column (stationary phase) is not cleaned properly?
Inadequate cleaning of the Reverse Phase HPLC column (stationary phase) results in a high back pressure, decrease in separation performance, broadening of peak, peak tailing and sometimes “ghost peaks” are detected.
This happens because of hydrophobic organic molecules, for example, lipids or large organic molecules easily stuck to RP HPLC Columns.
44. What is the procedure to clean the Reverse Phase HPLC column (stationary phase) quickly and efficiently?
Inject 100 to 200 microliter methanol or acetonitrile. Repeat this procedure 2 to 3 times. If a garbage peak still observes, do the normal flush with methanol or acetonitrile.
If you are using acetonitrile, ensure that it does not cause a precipitation of buffer-containing eluents.
45. What happens when the mobile phase is not degassed?
Noisy or drifting baselines and pressure fluctuations are signs of insufficient degassing.
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46. What are the various procedures used for mobile phase degassing?
• Refluxing
• Vacuum degassing
• Helium degassing
• Ultrasonic degassing
47. What are the suggested procedures for mobile phase degassing?
• Refluxing – Method is good but it is not practicable.
• Vacuum degassing – Method is good.
• Helium degassing – Method is good.
• Ultrasonic degassing – Ineffective method. Works good for acetonitrile/ water mixtures.
48. What are the advantages of acetonitrile in the mobile phase?
Low viscosity and better kinetics provides sharper peaks.
Acetonitrile/water mixtures have lower back pressure in comparison to methanol/water mixtures, hence, less wear and tear on seals and columns.
Higher elution strength, henace, lower solvent consumption. It provided similar elution strength at a lower concentration compared to methanol.
Silica gel is less prone to dissolve in acetonitrile compared to methanol (Reason: acetonitrile is less polar than methanol)
Low solubility of air, hence, less problems with air and easy for effective degassing.
Low UV absorptivity hence, better for detection at 195-200 nm
Better reproducibility, specifically while using ionic solutes because it causes small pH deviations in aqueous solutions.
Suitable for ion chromatography because it is a better solvating agent.
More toxic than methanol resulting in prevention of microbiological growth in the instrument.
Suitable for the separation of bases at lower pH and yield sharp peaks
49. What are the advantages of methanol in the mobile phase?
Methanol is odorless hence, it provides better working conditions.
Less toxic compared to acetonitrile.
Better solubility for salts hence, chances of precipitation low.
Methanol/water mixture brings the seals faster into its swelling conditions hence, equipment gets faster to working condition on aging of acetonitrile, impurities such as propionitrile, methacrylonitrile generate ghost'” peaks. This issue is less known for methanol hence, longer shelf life of methanol.
Good separation of bases at alkaline pH
Lower baseline noise above 220 to 230 nm
50. What could be the impact of too high or too low pH of the mobile phase on the C18 column?
Impact of high pH environment on C18 Column is silica gel dissolves at above pH 8. Because of
this, the column performance will rapidly decrease.
Impact of low pH environment on C18 Column, C18 chains are hydrolyzed at pH 2 and lower.
Because of that, the column bleeds, and the performance decreases.
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51. What is the solution to deal with very high (above pH 8) or very low pH (at pH 2 and lower) mobile phase?
Install a pre-column or old CI8 column between the pump and the injector.
On installation of this precalumn, the alkaline eluent will get saturated with silica gel and will not affect the separation column.
At low pH, the C18 chains of the pre column are hydrolyzed, protecting the main column.
52. What are the possible reasons for a change in retention time in HPLC analysis?
Following are potential reasons for change in retention time in HPLC analysis.
Change in stationary phase
Change in mobile phase
Change in temperature
Change in flow rate
Change in packing density
53. What are the most probable reasons for the short life span of the HPLC Columns?
Strongly adsorbed contaminants in the sample can ruin the column performance Shedding seals may clog column filters and the top layers of the packing.
Mechanical weakness of the packed bed which could be the consequence of rough handling of the column while handling in the lab or during shipment.
Inconsistent column life may occur because of adsorption of sample constituents on the top of the column. This could either occur because of precipitation because of low solubility of sample in the mobile phase or they may be strongly adsorbed.
This can also happen if one injects more samples. In that case, contaminants build up on the top of the column and prevent the sample to properly adsorb and distribute. This issue results in a peak distortion and an increase in back pressure.
Column collapsing may occur if a mobile phase pH outside the recommended range of column.
Sample dissolved in a strongly acidic or alkaline solution. Columns exposed to the wrong solvents.
Repeated column “washing”.
54. What are the solutions to increase the life span of the HPLC Columns?
Sample clean up with a suitable sample preparation method.
Use a guard column or precolumn. This will prevent column clogging and accumulation of contaminants at the top of the column. To have the highest performance of the guard column, use exactly the same packing as the analytical column.
(Not preferred method) Column washing could be one of the solutions that dissolves the contaminants on the top of the column. The drawback is washing will remove hydrolyzed bonded phase. Therefore, a repetitive washing may result in accelerated aging of the column.
(Not preferred method) Column backflushing. The drawback is when washing with a different solvent than the mobile phase, same consequences as column washing. While doing this using the mobile phase, it’s time consuming and sometimes it doesn’t work.
(Note: Backflushing should not be made as standard practice).
55. What are the possible reasons for variable Retention Times (RT) in HPLC analysis?
Randomly changing and inconsistent retention times – Pump(s) and the solvent mixing devices malfunction and not giving consistent flow.
Retention time does not change from run to run but they vary from day to day – most likely the source of variation is the composition of the mobile phase. Note: Rule of thumb, if you make an error of 1% in the amount of organic solvent, the retention time can change by between 5% and 15%, typically by about 10%.
Temperature fluctuations (Rule of thumb – RT change by about 1% – 2% per 1 ºC).
Air conditioning shut down usually at weekends.
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56. What are the solutions to prevent the variation in retention time in HPLC analysis?
Accurately preparation of the mobile phase. While preparing the mobile phase, measure the amount of solvent very carefully. Preferred method to prepare the mobile phase gravimetrically rather than volumetrically.
Method of degassing of mobile phase contributes to variability. Very good degassing method is applying vacuum and ultrasound together for about one minute.
Consequently there will be very good degassing with a nominal amount of solvent evaporation. Another good method is the use of helium sparging. Precaution: After initial equilibration, the flow of helium to be reduced to prevent helium to carry solvent vapors with it and reduce evaporation.
Accurate measurement of pH. A change of as little as 0.1 pH units can result in a retention time shift of 10%.
Control of the pH is critical when your sample contains ionic or ionizable compounds.
Temperature is an important factor to prevent the fluctuation of retention time.
57. What are the reasons for Drifting Retention Times in HPLC analysis?
Most common cause of drifting retention times is an equilibration problem.
In normal phase chromatography the Retention time is sensitive to the quantity of water adsorbed on the silica surface. Solubility of water in solvents such as methylene chloride or hexane is very low, column equilibration takes time. Still there are chances that RT my shift when dry hexane is used.
Equilibration in reversed-phase chromatography is quicker; 5 to 10 column volumes of mobile phase is generally sufficient to equilibrate column.
58. What is the solution for prevention of drifting Retention Times in HPLC analysis?
As we understood, the most common cause of drifting retention times is an equilibration problem.
In normal phase chromatography it is recommended to avoid using very dry solvents. Most preferable approach to solve the equilibration problem of silica with water is to use solvents that are “half-saturated” with water. It can be prepared by saturating a provided volume of
hydrophobic solvent with water and mixing it 1:1 with “dry” solvent. This will facilitate quick equilibration of the column.
In reversed-phase chromatography, 5 to 10 column volumes of mobile phase are generally good for quick equilibration.
59. What are the possible reasons for the peak shape issue of Peak Tailing?
Wrong mobile phase pH
Column void
Blocked frit
Unswept dead volume
Interaction with active silanols
Chelation with metal ions in stationary phase
60. What are the solutions to prevent peak shape issues of Peak Tailing?
Wrong mobile phase pH → Increase buffer concentration or Decrease mobile phase pH that suppress silanol ionization
Column void → Change the column, Column backwash
Blocked frit → Column backwash, Use inline filters
Unswept dead volume → Use shorter tubing connection and minimum number of connection, Ensure tightening of all connections
Interaction with active silanols or Chelation with metal ions in stationary phase → Add basic mobile phase additive or Use ultra-high purity silica based stationary phase