Hplc – Chemistry Interview

No Peaks
Tailing Peaks
Broad Peaks
Split Peaks
Extra Peaks
No Peak

Detector turned off- Check and make sure detector is turned off

Column to detector connection broken-

Ensure that column is properly connected to the detector

Ensure that there are no leaks between column and detector

No Sample Present

Make sure that the injector is not clogged and properly setup

Make sure the sample vial has enough sample ( Most softwares have injector setup to allow sampling from top/middle/bottom of the vial)

Make sure there are no air bubbles present( Need to rewrite/elaborate)

Make sure the sample material is not absorbed in the column or deteriorated at column temperature or conditions.

Tailing peaks
Void spaces in Column- Ensure that the column is properly packed,fill void/replace column as appropriate.
If the inlet bed of the column has dissolved, the particles become smaller and smaller until they drop down between the bigger particles, leaving a void at the top of the column and restricting the flow, giving rise to an increase in back pressure. Removal of the top fitting to the column may confirm that this is the case. The symptoms of a column void are broad tailing peaks and increased back pressure.

If there is a void, the normal procedure is to replace the column. Should you be on a desert island, or working in a lab where the lack of money makes you feel like you are on a desert island, you may like to try topping up the column with spare packing material. The easiest way to do this is to remove the top end fitting from the column, thereby exposing the void. As quickly as possible, transfer some wet packing material from the bottom of an old column of the same type, leaving the silica a little proud of the end of the column. Replace the inlet fitting on the column that had the void, preferably using a new frit. This should correct the poor peak shape, although the increased back pressure will remain.

Silica dissolves when the pH goes over 7, at elevated temperatures, in highly aqueous mobile phases, and at high buffer concentration. If your method uses any of these, then precautions need to be taken against silica dissolution, which can include:

Use a column which is stable at higher pH
Use a polymer-based column where there is no silica to dissolve
Use a pre-column before the injector to saturate the eluent with silicate anions.

Air bubbles in column- Most case air bubbles present in the column can be removed by reversing the column direction and flushing with an appropriate cleaning solvent. Refer to the column documentation to find proper procedures for column cleaning.

Ion Suppression Required- If the sample is an acid or base, the dissociation equilibrium between the ionised and unionised forms gives rise to very broad and sometimes tailing peaks. The solution is to use ion suppression. This involves adding an acid or base to the eluent to force the ionisation equilibrium to the unionised form. This means that for carboxyllic acids, an acid such as phosphoric acid is added (about 10-15ml/litre), and for bases, a base such as triethylamine or diethylamine is added, in about the same concentration.

Sample solvent a strong eluent-If the sample solvent is a strong eluent, in the first few seconds after injection, it drags the sample through the column, spreading out the bands, before it is diluted out of sight. This arises when difficulty is experienced getting an organic sample to dissolve in an aqueous eluent, and pure methanol or acetonitrile is selected instead. As a guide, the sample solvent should be no stronger an eluent than the eluent itself, and if using a gradient, than the gradient starting conditions.

Column Inlet Bed Contaminated- If the column inlet bed is becoming contaminated with components of the sample which bind strongly to the column, the active sites at the entrance to the column become blocked. This causes the sample to spread out, passing over the first particles in the column, and giving rise to broad and usually tailing peaks.

Column Overloaded- Use lower concetation sample or lower the injection amount. Larger diameter columns are also helpful.

Interfering/Co-eluting peak – Use longer column, Optimize one or all:mobile phase, column, flow rate, column temperature.

Wrong mobile phase pH– Properly Adjust pH. For basic compounds,lower pH usually provides more symmetric peaks
Broad peaks

Sample too large- Decrease sample size/Dilute sample

Co-eluting Peaks

Detector/Recorder Sampling rate too low- Increase sampling rate until satisfying peak is observed.

Detector time constant too low- Change detector time constant/peak width to match peak width

Incorrect tubing connection between the Injector-column/column- detector-The tubing used from the injector or autosampler and the column, and from the column outlet to the detector, should be of sufficiently fine bore that that sample passes through really fast. For analytical HPLC, working at around 1ml/min, this means using 0.010″ (0.25mm) id tubing or smaller. If working with PEEK tubing, either blue or red tubing would be good. Using a wider internal diameter will cause peak broadening.

Flow Cell Volume too large-This problem usually occurs when scaling down to narrow bore or microbore columns. If the flow cell volume becomes too large relative to the peak volume, the whole peak will fit in the flow cell at once and ends up being diluted, giving broad peaks, and poor peak heights. Usually this is seen immediately after scaling down, and hence it is relatively easy to diagnose. Also at the lower flow rates used with narrow bore columns, it is important to note that the id of the connecting tubing (see above) should be reduced proportionately. 0.13mm id is usually acceptable for tubing containing the sample.

Autosampler Wash Solution too Strong an Eluent-This is similar to the sample solvent point above, and applies to variable volume autosamplers. If the loop is say 200ul, and we inject 20ul, the remaining 180ul is filled with wash solution. If this is a strong eluent, the peak braodening effect described above for a strong sample solvent is amplified, because of the larger volume involved. Hence although the autosampler wash solvent must be strong enough to avoid sample carryover, it should not be a stronger eluent than the mobile phase, and in the case of gradient elution, it should not be a stronger eluent than the mobile phase at the gradient starting conditions.

Split peaks

Peak “splitting” is not characterized by any particular measurement; it is a subjective evaluation on the part of the chromatographer. Split peaks can generally be regarded as simply well-resolved shoulders. The causes and diagnostics follow similar rules.

If all the peaks in the chromatogram are split, and the problem appeared suddenly, then the following possibilities should be checked:

1. A partially-plugged frit at the column inlet.

2. A head space or void at the column inlet.

3. Incorrectly prepared mobile phase (pay special attention to buffer concentration and pH).

4. A dilution solvent that is stronger than the mobile phase.

If all the peaks in the chromatogram are split and the problem has been getting gradually worse, then the following possibilities should be checked:

1. A head space or void at the column inlet.

2. Chemical contamination or attack on the stationary phase. In most cases, the most cost-effective solution is simply to replace the column.

If only some of the peaks in the chromatograms are split, while other peaks look normal, and the problem appeared suddenly, then the following possibilities should be checked:

1. A dilution solvent that is stronger than the mobile phase.

2. Incorrectly prepared mobile phase (pay special attention to buffer concentration and pH).

3. (isocratic separations only) Check for extra-column volume (especially if early peaks tail more than later peaks).

4. Chemical contamination or attack on the stationary phase. In most cases, the most cost-effective solution is simply to replace the column.

5. A partially-resolved interference.

If only some of the peaks are split and the problem has gradually been getting worse, then the most likely cause is chemical contamination or attack on the stationary phase. In most cases, the most cost-effective solution is simply to replace the column.

Split Peaks can either be due to two similar peaks co-eluting or a single compound producing split peaks.

Extra Peaks

“Extra” peaks are almost alwyas real peaks, unless you are misinterpreting noise for a peak;

Most common reasons for extra peaks are:

Syringe not being cleaned properly between injections

Cross contamination between samples during sample preparation

Method time is not long enough for all the components to elute and thus there is a carry over from previous injection

Possible sources of contamination can include:
the water system
buffers/salts/additives
dirty glassware
contaminated pH electrodes, etc.
Column or instrument hardware

Prevention and care is the best approach for extra peaks problem

Hplc

Troubleshooting HPLC pressure

Posted on 27 May 201821 Feb 2019 by mahesh

Disconnect each part systematically from the HPLC system to check and see if you get a pressure drop and determine if the pressure built up is before or after the column

Pressure built up can be from several reasons:

Flow rate is set too high – Lower the HPLC pump flow rate

Dirty inlet frits/Inline filters-Replace frits/filters.

This problem can be identified by releasing the pressure valve on the inlet frits and see if you still observe high pressure. If all the pressure drops when the valve is open, frits are good. Move on to next step.

Next common place on the HPLC where the pressure can be built up is the needle/needle seat. Depending on your HPLC this will be different, Change your setting to bypass the needle and see if the high pressure drops, if yes, then the needle needs to be cleaned or changed.

Column temperature low or heater not turned on – Increase column heater temperature

It is possible to damage some columns by having high flow going through while at room temperture. Always make sure that the column temperature has reached operating temperature and stablized for ~30 minutes before raising the flow rate to operating flow rates.

Blocked guard column – Remove/replace guard column

Fluctuating Back Pressure

Bubble in Pump system or column- Thoroughly degas the solvent; Always only used 0.2U filtereted degassed solvents. Whenever possible use online degasser.

Leak in the Pump System- Most common places for the leaks are check valves, purge valves and any other coonections/seals. Check and make sure there are no leaks, Fix any leaks by tightening or replacing the appropriate part

Faulty check valve(s) – Replace check valve(s)

Using gradient elution – Pressure cycling is normal due to viscosity changes from the gradient.

Faulty Pump/injector system seal(s)- Identify the location and replace the seal(s)

In this case, you should note what normal fluctuation is for each gradient, to help idetify abnormal behaviou

Decreasing Back pressure

Insufficient flow from pump- Make sure the cap on the mobile phase reservoir is not too tight, that a vaccum is being created in the bottle thus making it difficult to pump mobile phase out of the bottle

Leak- Thouroughly check for leaks in pump systerm/ injector system and the column. You might see stable pressure at lower flow rate but low/decreasing pressure as you increase the flow rate. Some seals/connections might not be properly placed and leaks can form as system pressure increases.

Column temperature increasing- Column core temperature might not have stablized even if the controller shows a steady final temperature. Give enough time for column temperature to stablize. In rare instances, a faulty controller might be causing the column to overheat.

Increasing Back pressure

Blocked Flow lines- Systematically disconnect components from detector end to column end to find blockage; replace or clean blocked component

Particulate build up in column or pump- Always filter samples and mobile phase with 0.2U filter

Incompatible buffer/solvent system causing salt formation or precipitation- Ensure the solvent systems used are compatible

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Tailing peaks

Split peaks

Extra Peaks

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Fluctuating Back Pressure

Increasing Back pressure

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