Base line drift, Base line noise, Broad peaks, Change in height, Negative peaks, Ghost peaks in HPLC

Base line drift

 What You Expect                                                         What You Got

Disease	Medicine
Fluctuation of column temperature	Stabilize. Use column oven
Contamination of mobile phase/		Use HPLC grade solvent. Degas
Air in the detector cell	Clean cell. If necessary use a pressure restrictor at outlet
Plugged detector outlet line	Replace
Default mixing	Check mixer unit. Check flow rate and composition
Plugged detector outlet line	Replace
Strongly retained materials	Flush column with strong solvent
Unoptimized detection	Optimize detector

Base line noise

What You Expect                               What You Got

Disease	Medicine
Air (mobile phase, detector cell, pump)	Flush system, prime pumps, degas mobile phase
Pump pulse	Use a pulse damper
Incomplete mixing	Promote complete mixing
Electronic	Check electronic equipment in the same line
Leak	Please See No Peaks Or Very Small Peaks

 What You Expect                                 What You Got

Disease	Medicine
Leak	Please See No Peaks Or Very Small Peaks
Bad quality mobile phase	Use HPLC grade solvents
Electronics	Locate problem.Get servicing
Cell contaminant	Clean detector cell
Weak lamp	Replace detector lamp

Broad peaks

What You Expect                                 What You Got

Disease	Medicine
Altered mobile phase	Make new
Low flow rate	Increase
Leak	Please See No Peaks Or Very Small Peaks
Incorrect detector settings	Check and correct
Column overload	Please see Retention times are not constant
Void volume. Tubing too long or to wide	Use 0.010" tubing. Shorten path
Low buffer concentration	Increase
Column or guard column contamination	Replace
Void volume at inlet	Repack

Change in height

Disease	Medicine
Sample deterioration	Use fresh sample
Leak	Please See No Peaks Or Very Small Peaks
Non-reproducible sample volume	Ensure loop is completely filled
Low detector response	Check detector settings and operating conditions

Negative peaks

What You Expect                                 What You Got

Disease	Medicine
Recorder connections	Check polarity
Refractive index of mobile phase higher than that of solute	Change mobile phase
Vacancy peaks	Originate from great difference in composition between sample solvent and mobile phase. Dissolve sample im mobile phase
Mobile phase more absortive than sample components	Use mobile phase that is transparent at the wavelength used

Ghost peaks

What You Expect                                 What You Got

Disease	Medicine
Contamination of injector or column	Always flush injector after each injection.
Retained compound from previous injection	Flush colunm with strong solvent after operation to remove late eluting compounds

Reference:

Prof. Dr. H.J. Chaves das Neves

Universidade Nova de Lisboa

Peak tailing, Peak Fronting, Rounded Peaks in HPLC chromatogram

Peak tailing

What you expect                                   What you Got

Disease	Medicine
Active sites within the column	Test with standard test mixture. If OK add competing base or acid modifier
Wrong pH	Correct
Wrong column	Change
Void volume at inlet	May need repacking
Wrong sample solvent	Dissolve sample in mobile phase

Peak fronting

What you Expect                                What you Got    

Disease	Medicine
Column overload	See this (Retention Times are not constant)
Wrong pH	Correct
Sample solvent incompatible with mobile phase	See this (Peak Tailing)
Void volume at inlet	May need repacking
Wrong sample solvent	Dissolve sample in mobile phas

Rounded peaks

What you Expect                                  What you Got

Disease	Medicine
Detector outside linear dynamic range	Reduce sample
Gain too low	Adjust
Column overloaded	See this (retention times are not constant)
Time constants (detector, recorder) too high	Reduce
Wrong sample solvent	Dissolve sample in mobile phase

Reference:  
Prof. Dr. H.J. Chaves das Neves
Universidade Nova de Lisboa

Peak splitting

Peak splitting

What you Expect                                   What you Got

Disease	Medicine
Contamination of column or guard	Remove guard column. If the problem is solved replace it. If not go next
Plugged column	Use adequate recovery procedure. If the problem persists go next
Plugged inlet frit	Replace. If the problem persists discard column

Retention times are not constant

Retention times are not constant

What you Expect                                 What you Got                                         

Disease	Medicine
Leak	Check for loose fittings Check pump for leaks, salts and noise Check seals
Change in mobile phase composition	Check. Make new
Air trapped in pump	Check flow Prime pump. Check and change seals. Be sure the mobile phase is degasse
Temperature fluctuations	Stabilise column temperature. Use column oven
Overloading	Dilute sample
Sample dissolved in a solvent that is incompatible with the mobile phase	Dissolve sample in the mobile phase whenever possible. Adjust

Reference: 

Prof. Dr. H.J. Chaves das Neves

Universidade Nova de Lisboa 

No Pressure or Pressure Lower than anticipated

No Pressure or Pressure Lower than anticipated

                     

What you Expect.                                 What you got

Disease	Medicine
Leak	Check for loose fittings Check pump for leaks, salts and noise Check seals
Mobile phase interrupted	Check reservoirs. Check position of the inlet tubing Check loop for obstruction or air Check degasing of mobile phase Check compatibility of the mobile phase components
Air trapped in pump	Check flow disconnect column and prime pump Flush system with 100% methanol or iso propanol Contact servicing if necessary
Worn pump seals	Replace seals Check pistons and replace if necessary

Higher Pressure than anticipated

  What you Expect.                                 What you got

Disease	Medicine
Pump, injector, tubing	Disconnect column, run pump at 25 mL/min: Is the pressure minimal? Go to next step If pressure still high check systematically from detector to pump for obstruction
Obstructed column or guard	If guard column is obstructed, replace it If the analytical column is obstructed reverse it and flush disconnected from the detector. Use adequate restoration procedures Change inlet frit Replace de column

            

reference: 

Prof. Dr. H.J. Chaves das Neves

Universidade Nova de Lisboa 

No Peaks or view Small Peaks in HPLC

No Peaks or view Small Peaks in HPLC

What You Expect                                    What You got

Disease	Medicine
Detector off	Check Detector
Broken connections to recorder	Check connections
No flow	Check flow
No sample/Wrong sample	Check sample. Be sure it is not deteriorated. Check for bubbles in the vials
Wrong settings on recorder or detector	Check attenuation. Check gain

No Flow

Disease	Medicine
Pump off	Start pump
Flow interrupted	Check reservoirs. Check position of the inlet tubing Check loop for obstruction or air Check degasing of mobile phase Check compatibility of the mobile phase components
Leak	Check fittings Check pump for leaks and precipitates Check pump seals
Air trapped in the system	Disconnect column and prime pump Flush system with 100% methanol or isopropanol Contact servicing if necessary

Reference:
Prof. Dr. H.J. Chaves das Neves
Universidade Nova de Lisboa