61. What are the possible reasons for the peak shape issue of Split Peaks?
Contamination on column inlet
Incompatibility of Sample solvent with mobile phase
Elution of second component simultaneous
Blocked frit
Column overloaded
62. What are the solutions to prevent peak shape issues of Split Peaks?
Contamination on column inlet →
1. Use guard column, or replace guard column, or replace analytical column (depending on issue identified),
2. Backwash analytical column (less preferred method and should be rarely used)
3. If contaminants are strongly adsorbed, try to regenerate column Or 4. Replace column if issue is not resolved
Incompatibility of Sample solvent with mobile phase →
Elution of second component simultaneous →
1. Sample cleanup before injection,
2.Modify mobile phase composition, or
3. Change stationary phase depending on selectivity
Blocked frit → Use in-line filter, Column backwash
Column overloaded →1. Increase column capacity by using high capability stationary
phase or increase the dimension, or, 2. Decrease injection volume
63. What are the possible reasons for the peak shape issue of Peak Fronting?
Sample solvent incompatible with mobile phase
Formation of channels in column
Low temperature of column oven
Column overloaded
64. What are the solutions to prevent peak shape issues of Peak Fronting?
Low temperature of column oven → Increase column oven temperature to increase column temperature
Column overloaded → Use less injection volume or dilute the sample solution according to desired column load
Formation of channels in column → Refer to the column literature to operate the system
within the given range of parameters, e.g. pH of the solution. If problem persist, replace the column
Sample diluent incompatible with mobile phase → Change the sample diluent as mobile phase
65. What are the types of Retention Time Variation or RT Variation?
Decreasing Retention Times
Increasing Retention Times
Fluctuating Retention Times
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66. What are the probable reasons for Decreasing Retention Times?
High flow rate
Column overloaded
Loss of bonded stationary phase
Active groups on stationary phase
Incorrect column selection
67. What are the solutions to prevent the issue of Decreasing Retention Times?
High flow rate – Check and regulate the flow rate of pump
Column overloaded – Decrease sample injection volume, Select the column with larger internal diameter
Loss of bonded stationary phase – Replace column, Operate at recommended pH range for RP columns. Generally operated at 2-8 for silica based RP HPLC.
Active groups on stationary phase – Increase buffer strength
Incorrect column selection – Select the column with larger internal diameter
68. What are the probable reasons for Increasing Retention Times?
Changing mobile phase composition
Low flow rate
Loss of bonding in stationary phase
Bubbles in mobile phase
69. What are the solutions to prevent the issue of Increasing Retention Times?
Changing mobile phase composition – 1. Cover solvent reservoirs, 2. Prepare fresh mobile phase
Low flow rate – 1. Check and adjust pump flow rate, 2. Check for leaks in system, including pump seals
Loss of bonding in stationary phase – Replace column
Bubbles in mobile phase – 1. Check flow rate and pressure, 2. Degas mobile phase
70. What are the probable reasons for Fluctuating Retention Times?
Inadequate column equilibration
Mobile phase preparation error/ composition variation
Inadequate buffer capacity
Unstable column temperature
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71. What are the solutions to prevent the issue of Fluctuating Retention Times?
Unstable column temperature –
1. Check column oven to ensure temperature stability,
2. Malfunctioning of the column oven thermostat
Inadequate column equilibration –
1. Allow columns to equilibrate for sufficient time between runs,
2. Condition the column using concentrated sample
Mobile phase preparation error/ composition variation –
1. Verify volume make-up of mobile phase, if needed, prepare fresh one,
2. Verify the proportioning-valve accuracy
Inadequate buffer capacity –
Utilize the buffer concentrations more than 20mM
72. What are the probable reasons for Ghost Peaks?
Contamination of column
Contamination of injector
Carryover/ late eluting peak from previous injection
Contaminated water or solvent
Method specificity not established adequately
73. What are the solutions to prevent the issue of Ghost Peaks?
Contamination of column –
Adequate column washing/ Flushing of column to remove contaminants
Contamination of injector –
Injector flushing between analyses
Carryover/ late eluting peak from previous injection –
1. Increase the run time,
2. Flush column with strong mobile phase at end of each run,
3. For gradient runs, end the run at higher concentration
Contaminated water or solvent –
Use HPLC grade water or solvent
Sample contamination –
Ensure cleanliness of glasswares, sample preparation aids such as mortar and pestle, cleanliness of sample storage area
Method specificity not established adequately –
Use sample clean-up process and
perform method specificity covering all the relevant factors (excipient peak interference, degradants, etc.)
74. What are the probable reasons for Negative Peaks?
When using RI detector, Refractive Index of solute is less than the mobile phase When using UV detector, solute absorption is less than absorption of mobile phase Different composition of Sample solvent and mobile phase
75. What are the solutions to prevent the issue of Negative Peaks?
When using the RI detector, the Refractive Index of solute is less than the mobile phase –
1. Use mobile phase with less refractive index,
2. Invert detector polarity to get positive peaks
When using a UV detector, solute absorption is less than absorption of mobile phase –
1. Change UV wavelength to get a positive peak,
2. Identify mobile phase so that it lowers the UV absorption
Different composition of Sample solvent and mobile phase –
Revise the composition of either sample solvent and or mobile phase to improve the compatibility and sample solubility
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76. What are the probable reasons for Spikes in a chromatogram?
Air bubbles in mobile phase/ inadequately degassing of mobile phase
Column stored without closing ends using column closure caps
77. What are the solutions to prevent the issue of Spikes in a chromatogram?
Air bubbles in mobile phase/ inadequately degassing of mobile phase –
1. Degas mobile phase using suitable method,
2. Use back pressure restrictor at detector outlet, 3. Ensure all tubings are without any leaks and fittings are tight
Column stored without closing ends using column closure caps –
1. Ensure that column is stored with end caps closed,
2. Flush Reverse Phase column using degassed methanol
78. What are the probable reasons for High Back Pressure in HPLC systems?
Wrong HPLC pump setting
Pressure higher during middle of gradient
Temperature lower than required
Column ageing
Column frit blockage
In-line filter blockage
Guard column blockage
System blockage
Buffer precipitation in the system
79. What are the solutions to prevent the issue of High Back Pressure in HPLC systems?
Wrong HPLC pump setting – Verify HPLC pump setting and adjust appropriately
Pressure higher during middle of gradient – This is normal phenomenon
Temperature lower than required – Maintain the column oven temperature as per method requirement
Column ageing – It is normal phenomenon of gradual increase in pressure over the period of lifetime
Column frit blockage – 1. Column backwash, 2. Use an in-line filter to prevent column
blockage and reduce backpressure, 3. Ensure efficient filtration, 4. Use guard columns
In-line filter blockage – 1. Replace in-line filter frit with new one, 2. Centrifuge or filter samples with recommended filters, 3. Degas and Pre-filter mobile phase
Guard column blockage – Increase the replacement frequency depending on experience
Buffer precipitation in the system – Back flush the column using water
80. What are the probable reasons for Low Back Pressure in HPLC systems?
Leakage in the HPLC system
Column temperature is higher than required
Flow rate is lower than required
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