3-problems encountered during the analysis, their causes and solution

Now a chromatographic method ( the appropriate combination of stationary phase,and flow rate) which gives us a satisfactory chromatogram has been chosen. Several samples of the same mixture were analized and a reproducible pattern was obtained. The analysis should be running smoothely afterwords to enable us to construct a calibration curve and analize our unknown samples. Sometimes, however, we may run into unexpected problems (changes in performance of the instrument or changes in the chromatographic pattern that has been reproducible thus far etc...) which should not normally happen. Finding out the causes of the problem and fixing them is a valuable experience a good chroamtogrammer should acquire. There are several books and manuals dealing with the subject (truoble shooting). However, here are some of the problems we commonly face, their possible causes and solitions.

I- No peaks or very small peaks

Chromatogram(A)

Chromatogram(B)

The two chromatograms given above show the normally produced chromatogram (A) which was a good chromatogram then we run into a problem where we see no peaks coming out of the colum. A couple of causes (problems) might have arrised which lead to this.

Proble1em(1)

No peaks or very small peaks	Possible cause	solution
	Detector is off ( forgot to turn on the detector).	Check detector
	Broken connections to recorderelectric connections theat carry the signals to the detector and from the detector to the recorder are broken).	Check detector connections
	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 wavelength setting (wrong wavelength) and sensitivity setting

Problem (2)

No Flow	Possible cause	solution
	a-Pump off	Start Pump
	b-Flow interrupted	Check reservoirs. Check position of the inlet tubing. Check loop for obstruction or air. Check degassing of mobile phase. Check compatibility of the mobile phase components
	c-Leak	Check fittings. Check pump for leaks and precipitates. Check pump seals.
	d-Air trapped in the sys	Disconnect column and prime pump. Flush system with 100% methanol or isopropanol

II-CHanges in pressure

Problem (3)

Decreasing Pressure( no pressure or lower than usual	Possible cause	solution
	Insufficient flow from pump	Loosen cap on mobile phase reservior (tigt capping of the mobile phase container while the pump is pulling solven at high rate may create vacume thus preventing the pump from pulling out the right ammount of solvent).
	Leak in hydraulic lines from pump to column.	Check reservoirs. Check position of the inlet tubing. Check loop for obstruction or air. Check degassing 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

Problem (4)

Increased pressure(pressure higher than usual)	Possible cause	solution
	Column blocked with irreversibly adsorbed sample	Improve sample cleanup; use guard column; reverse-flush column with strong solvent to dissolve blockage.
	Microbial growth	Use at least 10% organic modifier in mobile phase; use fresh buffer daily; add 0.02% sodium azide to aqueous mobile phase; store column in at least 25% organic solvent without buffer.
	Salt precipitation (especially in reversed-phase chromatography with high concentration of organic solvent in mobile phase).	Ensure mobile phase compatibility with buffer concentration; decrease ionic Ensure mobile phase compatibility with buffer concentration; decrease ionic Check fittings. Check pump for leaks and precipitates. Check pump seals
	The pressure comes back to normal when the injector is disconnected from column = blockage in injector.	Clean the injector
	Blocked column frit.	1. Backflush column (run the solvent opposite to the normal direction of flow (if permitted)). 2-Replace the frie

III-Change in the chromatogram

a-Problem(5)

Variable retention time (Change in retention time)

Retention time of the different peaks of the mixture sgould be reproducible as long as there was no change in the chromatographic conditions (mobile phase, stationary phase or flow rate). In fact some times retention time is used as a confirmation of the structure of an unknown peaks.In the given chromatograms, the three peaks come out at 1.5, 4 and 7 minutes respectively. after several reproducible runs, without intentional changes in conditions, the peaks started coming out farther apart at 6, 10 and 15.

Variable retention time (Change in retention time)	Possible cause	solution
	Contamination buildup	Flush column occasionally with strong solvent.
	Selective evaporation of mobile-phase component.	Cover solvent reservoirs; use less-vigorous helium purging; prepare fresh mobile phase.
	Loss of bonded stationary phase.(Column aging)	Some times adjusting pH of mobile between 2 and 8 may help. Or change the column.

b-problem(6)

peak tailing

Ipeak tailing	Possible cause	solution
	a-Blocked frit	1. Reverse flush column (if allowed)2. Replace inlet frit.
	b-( not all peaks ar tailing) interfering peak(s)	1. Use longer column 2. Change mobile-phase and/or column (selectivity)
	c-Something wrong with the mobile-phase pH	1. Adjust pH 2. adjust components of mobile phase.
	d-Sample reacting with active sites.*	1.Add ion pairing reagent (an ion that forms a pair with the compound being eluted thus preventing it from reacting with the active sites on the selica). 2.Change the column.

*explanation: when the column is used for a long time some of the chemically bonded substances (e.g. C18) may be depleted exposing active groups on the selica (aging) which bind strongly to some of the components of the mixture causing them to be eluted with difficulty and thus dragging behind the main bulk of the peak causing a tail formation.

problem (7)

Split Peaks

Split Peaks	Possible cause	solution
	a-Contamination on guard or analytical column inlet.	1. Remove guard column and attempt the analysis 2.Replace guard column if necessary 3.If analytical column is obstructed, reverse and flush. 4.If the problem persists, then the inlet is probably clogged. Change frit.
	b-Sample solvent incompatible with mobile phase	Change solvent; and whenever possible, inject samples in mobile phase

problem (8)

The appearance of extra peak (s)	Possible cause	solution
	a-contaminating components in the sample	clean up the sample.
	b-Late eluting peak(s) :peaks which did not come out during the previous analysis and started coming out after the next sample was injected.	Increase run time or gradient slope (increasing gradient slope leads to faster change in solvent composition and therefore better resolution).

problem(9)

Base Line drift

Base Line drift	Possible cause	solution
	a-Mobile phase contaminated, deteriorated, or prepared from low-quality materials.	Use HPLC-grade solvents, high-purity salts, and additives.
	b-Contaminant or air buildup in detector cell.	Flush the cell with methanol or other strong solvent.
	c-Mobile-phase mixing problem or change in flowrate.	Correct composition of the mobile phase or flow rate (to avoid running into such a problem, routinely monitor composition and flow rate).
	d-Slow column equilibration, especially when changing mobile phase	Run 10–20 column volumes of new mobile phase before analysis

problem (10)

Baseline Noise

Baseline Noise	Possible cause	solution
	a-Air in mobile phase, detector cell, or pump.	1. Degas mobile phase 2. Flush to remove air from detector cell or pump.
	b-Other electronic equipment on the same line.	Isolate detector, or recorder to determine if the source of problem is external; correct as necessary.
	c-Pump pulsation.	Incorporate pulse dampener into system.

problem (11)

broad peaks

broad peaks	Possible cause	solution
	a-Mobile-phase composition changed.	Prepare new mobile phase.
	b-Mobile-phase flow rate too low.	Adjust flow rate.
	c-Leaks (especially between column and detector)	1. Check for loose fittings 2. Check pump for leaks, salt build-up. 3. Change seals if necessary.
	d-Column overloaded	Inject smaller sample on the column (e.g., 10 µl vs.100 µl) or 1:10 and 1:100 dilutions of sample.
	e-tubing between column and detector too long or ID (internal diameter) too large	use shorter piece of tubing or tubing with smaller ID
	f- guard column contaminated or worn out.	replace guard column.
	g-Column contaminated or worn out	flush old column with strong solvent or replace the old column with a new one.
	h- peaks represent two or more poorly resolved compounds (loss of resolution)	see the next problem.

problem(12)

Loss of resolution

loss of resolution	Possible cause	solution
	a-mobile phase contaminated or deteriorated (causing retention times to change)	prepare new mobile phase.
	b-obstruction of gaurd or analytical column.	1- remove gaurd column and attempt the analysis and replace the gaurd column if necessary 2- if the analytical column is obstructed reverse and flush if the problem pesists then the column may be fouled with strongly retained contaminant or the inlet may be blocked then replace the frit or replace the analytical column
	c-Aging or deteriorated column (will lead to loss of selectivity)	Replace the column with a new one