28/04/2019 Common Peak Shape Distortions in HPLC and their Prevention

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POSTED ON JANUARY 17, 2014 BY DR. DEEPAK BHANOT 9 COMMENTS

Common Peak Shape Distortions

in HPLC and their Prevention
A chromatographer always looks forward to getting perfect shaped peaks for each and every
analysis but in reality peaks get distorted due to numerous reasons. Distortions are frustrating
but if proper corrective steps are taken peak shape distortions can be avoided.

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A at top peak response arises when the detector gets overloaded with the sample. In such case
Gas Chromatography (55)
detector sensitivity is much higher and it gets saturated to give a broad at top peak. One option
is to attenuate the signal response (not a practical solution) as detection of other peaks will also General Topics (140)

get a ected. A more viable option is to reduce the concentration of sample or volume of Guest Posts (27)
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Tailing Peak
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Peak tailing is a common distortion arising due to ionization of surface silanol groups to – Si-O –
Stability studies (8)
which provide cationic exchange sites. The solution lies in use of high purity grade silica support
along with a selection of the recommended pH range 2-8. Training (21)

Weighing (3)
Use of bu er control additives also limits ionization of silanol groups on the silica surface.
Additives such as Tri uoroacetic acid (TFA ) an ion pairing agent helps suppress ionization. Bu er
additives in the concentration range 10– 25 mM are su cient for most applications.

Frit blockage or void formation can be prevented by ltering mobile phase solvents, use of inlet
lters and replacement of pump seals before they begin to wear and release garbage particles.

Low purity silica often contains metal impurities which promote ionization of silanol groups. Use
of high purity silica stationary phase helps in tailing due to chelation with metal ion in stationary
phase.

Peak Fronting

Fronting Peak

Peak fronting is less common in comparison to Peak tailing

Generally peak fronting as a result of channeling inside the column. It is best to replace the
column or otherwise operate within the recommended pH limits.

Column overload can also result in peak fronting. Use dilute sample or reduce the sample
injection volume.

Incompatibility of mobile phase with sample often results in peak fronting. Dissolve sample in
mobile phase or another compatible solvent

Peak Shoulders and Split Peaks

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28/04/2019 Common Peak Shape Distortions in HPLC and their Prevention

Split peak
Shoulder Peak

Shoulder peaks and split peaks often result due to presence of two closely unresolved
compounds.

Reduce sample size or use a diluted solution often resolves the split

Splitting o peaks is also caused by frit blockage. Reverse ow with 20 – 30 ml of mobile phase
often resolves the peak splits.

Replace the blocked frit or the guard column

When split is caused due to presence of two closely eluting compounds use sample cleanup prior
to injection

As you have seen above a number of causes of peak shape distortions are common such as pH
control of mobile phase, blockage due to particulate contamination, blocked frits and column
overload. The shape of the distortions can be improved by taking corrective steps and other
recommended steps whenever necessary.

Please share your experience and leave your comments.

Filed Under: HPLC Tagged With: Chromatography, High-performance Liquid Chromatography, Hplc,
Peaks

About Dr. Deepak Bhanot

Dr Deepak Bhanot is a seasoned professional having nearly 30 years expertise
beginning from sales and product support of analytical instruments. After
completing his graduation and post graduation from Delhi University and IIT
Delhi he went on to Loughborough University of Technology, UK for doctorate
research in analytical chemistry. His mission is to develop training programs on analytical
techniques and share his experiences with broad spectrum of users ranging from
professionals engaged in analytical development and research as well as young enthusiasts
fresh from academics who wish to embark upon a career in analytical industry.

Comments

Deepak Pandey says:

January 28, 2014 at 8:00 pm

Dear sir give your e-mail id. I have some query, hope you have answer for that.

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28/04/2019 Common Peak Shape Distortions in HPLC and their Prevention

Reply

Ramesh Vaze says:

April 21, 2016 at 8:09 pm

Hi Deepak
Nice information on HPLC peak
I am M Sc Analytical Chemistry (Mumbai University 1985)Institute of Science
I was Working for Varian India As Product Specialist
Now Running my own Business as Scienti c Instruments selling Refurbhised HPLC
GC HS AAS in Mumbai
I have Sold TSP FL 3000 to one cutomer in Nasik for Agrochemcal Analysis by HPLC
We are grting lots of noise and Humps when we injected Dervistised Sample of
Garpe Extract
What could be Problem
The Detector is Hooked up to waters Binary System to Empower II through SAT in
Bpox
We are Geting Nosie

Reply

Dr. Deepak Bhanot says:

April 22, 2016 at 5:23 pm

Hi Ramesh,
During the derivatization process it appears that some impurities are getting
introduced . As unidenti ed impurities are the main contributors to noise try
using derivatization reagent of higher purity.Also take care to degas the mobile
phase thoroughly as minute air bubbles can also give rise to random noise.

Reply

zak says:
February 25, 2014 at 4:42 am

Dear sir give your e-mail id. I have some query, hope you have answer for that

Reply

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28/04/2019 Common Peak Shape Distortions in HPLC and their Prevention

Dr. Deepak Bhanot says:

February 25, 2014 at 12:34 pm

My mail id deepak.bhanot@aurigaresearch.com

Reply

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Dr. Deepak Bhanot says:

December 8, 2017 at 10:24 am

No laid down rules for blogging. Simply put yourself in the shoes of your targeted
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