2006md 7 - troubleshooting

8/7/2019 2006MD 7 - Troubleshooting

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HPLC

Troubleshooting

A.NARENDER

Asst.Service Manager

Spinco Biotech Pvt Ltd.

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Daily Maintenance

Pre-analysis checks (1)

Filter mobile phase

Wait for mobile phase to reach room temperature

Purge all the flow lines with mobile phase

Replace mobile phases as necessary

If buffer is used as mobile phase, wash the back of plungerseal

If autosampler is used, verify that the autosampler rinse bottle is

full

Purge the autosampler rinse solution

Check for leaks

Check the pump pressure

Check the oven temperature

Perform a baseline check


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Daily Maintenance

Post-analysis checks

Wash the column

If the column is not going to be used for long time, take off it

from the system after washing, and cap both ends of the

column, store it in cool and dark place

Clean the flow line or the rest of the instrument, as necessary

Store the solvent reservoir filters in rinse solvent to prevent

them from drying out

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Outline

- Troubleshooting Process

- Categories of Problems

Pressure Issues

Baseline Issues

Peak Shape Issues

Retention Issues

- Troubleshooting Examples


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Troubleshooting Process

Gather the facts not theories.

Compare the performance obtained to the expected performance.

List possible causes.

Check the simplest things first its easier.

Work through the possible causes in a step-by-step manner

checking the outcome from any changes made.

As a last resort get help from elsewhere, for example your

instrument supplier help desk or your local technical support

department.

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Potential Sources of Problems

Mobile phase

Injector

Pump

Sample

Column and guard column

Detectors

Tubing and connectors

etc.

Analysts should simplify the problem source

chemistry or mechanical?


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Troubleshooting Guide

Main Troubles with HPLC1. Pressure

Low pressure

Pressure fluctuation

High pressure

2. Unstable Baseline

Drift/noisy

Spikes

Sawtooth

Cyclic

No change

3. Distorted Peak Shape

Broading

Tailing

Leading

Splitting

No peaks

4. Retention Time

Changing Retention

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Pressure Low pressure

Leaking in pumps, check

valves, seals or connectors

Pump cavitation

No pumping


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Possible causes :

1. Air bubbles in pump head

2. Leaks in system

3. Check valvesare dirty

4. Plunger seal is damaged

5. Plunger is spoilt.

Remedy:

1. Purge the pump2. Check for leaks in all the connections, especially new connection.

3. Clean check valves in ultrasonicator

4. Replace plunger seal

5. Replace plunger

Pressure Fluctuation in System

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High Pressure in System

Possible Causes

Clogging in

Detector cell Column

Pump

Mixer

Piping

Too much work ?

Too much nagging ?

Medical problem ?


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Locate the point of the pressure increases

Column or system?

Check the system methodically:

Disconnect column from detector, if the pressure problem goes way,

the problem is clogging of detector cell or piping to and from flow

cell.

If pressure persist, disconnect guard column and main column. If

pressure becomes normal, clean/replace main column.

If pressure persist, remove guard column. If pressure is normal,replace guard column.

Continue in this manner from auto-injector (if present) to mixer and

finally to pump until find out the high pressure spot.

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Possible reason:

Clogging in system. It may occur in any part of system.

Remedy :

If clogging in piping, it may be possible to flush out using

IPA or nitric acid at higher flow rate. In this case, do not

connect column or detector.

Clean or replace in-line filter.

High Pressure in System



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Possible reason:

Column clogging/failure.

Accumulation of particulates and/or adsorbed substances on column

Remedy :

1.Clean guard column and column.

2.Replace filter frit on column if possible.

3.Flush the column reversely or repair the column (This is recommended

only as a last resort).

Precaution:

In these cases, it is always recommended to use guard column

whenever

possible. The guard columns are cheaper and can be easily replaced

when clogging or adsorption occurs.

High Pressure in Column


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Precautions for Column

Buffer solution must be filtered

by 0.2 or 0.45 m membrane filter.

Sample must be filtered

by 0.2 or 0.45 m membrane filter.

Be sure do not jump from buffer to pure organic or from organic tobuffer. This can lead to buffer precipitation, plugging and pressureproblems.

Always use a washout, intermediate solvent.


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l ti (1)

Wash the column with suitable solvent

Open the column end, carefully remove the

filter/frit with the column in an upright

position, and sonicate it.

Change the filter/frit.

Wash the column reversely at low flow rate.

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C l m Clea i g

Washing the column will remove non-polars.

Buffered mobile phase:

must wash out the buffer with the same mobile phase minusthe buffer first

Aqueous organic solvent

Wash with Acetonitrile or methanol for at ~20 column

volumes.


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Column Cleaning

For reversed phase column

1. Wash with mobile phase without buffer salts

2. Wash with methanol or acetonitrile

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Solution (2)

only ~1 cm might be polluted

column

Last resort:

Remove the contaminated part and

repack with clean packing material.

Always use the same size and type of

material used originally in the column.


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Baseline Troubleshooting

Noisy baseline

Sawtooth

Drift

Cyclic

Spikes

Positive & negative peaks


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Unstable Baseline

Contaminated column

Detector cell is dirty

System is contaminated

Weak detector lamp

Leaks

Unstable pumping

Mobile phase is not degassed properly

Air bubbles in pumps/detector

Electronic noise

Temperature is fluctuating

Wavelength is too low

More

en? Why is itnot flat?


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Spike Baseline

Bubbles in flow cell

Apply back pressure to the flow cell

Degas mobile phase

Clean flow cell with IPA

Pump pulsation

Check pump function

Install damper

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Baseline - Sawtooth

Bubbles

Mixing problem

Plugged lines

Electrical problems


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Temperature fluctuations

Mixing problem

Mobile phase was not degassed

Pump problem

Baseline - Cyclic

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Baseline no change

Faulty circuits

replace any faulty parts

Lamp is off

set lamp parameter to turn on lamp power

Range setting is not correct

enter an appropriate range value


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Peak Shape Issues

Peak broading

Peak tailing

Peak fronting

Peak splitting

Other asymmetry peaks

No Peaks

Many peak shape issues are combined


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Peak Broading

Sample overload

Large extra system volume

Retention time is too long

Poor column efficiency


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Peak Tailing

T e fl -line ntain e tra ea l e

a le erl a ing

ili a- a e l n egra e at ig r ig

te erat re

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Peak Fr nting

a le erl a ing

C anneling in l n


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Sample is too diluted

Injection volume is too small

Detector lamp is off

Leaks

Pump is not pumping

Injector is damaged

Zoom setting of chromatogram is not correct

Column retains all compounds

Incorrect mobile phase/sample

o Peaks

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Changing Retention Time

Contamination was built up

Equilibration time is insufficient

Inconsistent mobile phase preparation

Inconsistent on-line mobile phase mixing

Selective evaporation of mobile phase component

Unstable column temperature

Leakage

Unstable pumping



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Tips

Good habits

Log book record pressure and mobile phase

Wash the column after sample analysis

Read manual/instruction

If any problem happens

Observe the instrument carefully look, smell, and feel

Check the simple things first

The hardware is not easy to be broken

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Thank You!

2006md 7 - troubleshooting

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