28/04/2019 How to Deprotenize Biological Fluids prior to HPLC analysis?

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POSTED ON JANUARY 25, 2014 BY DR. DEEPAK BHANOT LEAVE A COMMENT

How to Deprotenize Biological

Fluids prior to HPLC analysis?
Deproteinization has contributed to the ever-expanding scope of HPLC analysis of biological
uids. Sample ltration before HPLC analysis. Importance of sample ltration in liquid
chromatography analysis is strongly recommended to extend column life and reduce
maintenance costs thereby contributing to increased laboratory throughputs.

In addition to ltration, deproteinization is necessary for analysis of biological uids such as

blood plasma, serum, urine and cerebrospinal uid for levels of antibiotics, residual pesticides
and psychotropic drugs. Proteins need to be precipitated and removed from the sample
otherwise these can contribute to the deterioration of column performance

Deproteinization
Techniques
Denaturation – heating denatures most
proteins and after heating denatured
particles are centrifuged and removed.

Chemical precipitation – more e cient than POPULAR LATEST

heating as temperature sensitive

TODAY WEEK MONTH ALL
compounds can deteriorate in the heating
How to Deproteinized Biological Fluids prior to Jetpack plugin with Stats module
process. Acidi cation with trichloroacetic
HPLC analysis needs to be enabled.
acid, centrifugation, and neutralization with
sodium hydroxide removes 99% of the
proteins. Perchloroacetic acid can also be
used. Both the acids have some drawbacks. Trichloroacetic acid absorbs strongly below 230 nm TOPICS
whereas perchloroacetic acid leaves salts in solution which can precipitate with organic solvents
or contribute to baseline noise of UV detector AAS (53)

Analytical Chemistry (3)

Acetonitrile treatment – acetonitrile is a common solvent transparent below 190 nm. Mixing and
Calculations (5)
centrifugation of plasma sample and acetonitrile will lead to precipitation of about 95% of
proteins. Non-polar proteins such as albumins remain in liquid phase. The supernatant can be Chemical (2)
injected directly if a guard column is used to remove the balance 5% of the proteins. On long-
Chromatography (9)
term use, the guard column will need to be replaced or inverted and washed out to prevent the
proteins from entering the main column. Environmental Analysis (2)

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On-line Deproteinization Free e-courses (2)
Generally, it is su cient to carry out the deproteinization o -line followed by centrifugation after
Gas Chromatography (55)
adding an acid or organic solvent. However in order to save time automated on-line
deproteinization is desirable. Several instrument suppliers o er dedicated accessories which General Topics (140)

consist primarily of a pre-treatment column: packed with the hydrophilic polymer coating. Guest Posts (27)
Macromolecular proteins are retained whereas the analytes are led to the main column.
HPLC (60)

We have seen that the deproteinization has expanded the applications of HPLC separations of ICP Techniques (13)
biological uids which are complex matrices and HPLC estimations on such uids would have
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been di cult if not impossible without using the technique.
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Please share your views and leave comments based on your experiences. Lab Management (95)

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About Dr. Deepak Bhanot Microbiology (14)

Dr Deepak Bhanot is a seasoned professional having nearly 30 years expertise
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beginning from sales and product support of analytical instruments. After
completing his graduation and post graduation from Delhi University and IIT Paper Chromatography (10)
Delhi he went on to Loughborough University of Technology, UK for doctorate research in
pH Measurement (3)
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 Preformulation Studies (3)
analytical development and research as well as young enthusiasts fresh from academics who
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wish to embark upon a career in analytical industry.
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