1) Hydrogels created through molecular imprinting have the potential to selectively filter metabolites like glucose from biological samples, improving quantification of other molecules by GC-MS metabolomics.
2) Preliminary studies show poly(allylamine) hydrochloride hydrogels imprinted with glucose-6-phosphate are selectively binding for glucose over fructose.
3) Future work will examine imprinted and non-imprinted hydrogel binding capacities for metabolites in standard solutions and complex biological mixtures to explore their utility in metabolomics analysis.
Affinity chromatography is a method of separating biochemical mixture based on a highly specific interaction between antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid.
Affinity chromatography by Shiv kalia ( m.pharma analytical chemistry)Shiv Kalia
Detailed introduction of (Chromatography and Affinity Chromatography) and its theory, principle ,working ,application and limitation of Affinity Chromatography . This chromatography technique is also useful for GPAT ,UGC NET , GATE, DBT aspirants.
It includes basic knowledge about affinity chromatography along with its procedure and application. and brief description of various type of affinity chromatography.
Affinity chromatography is a method of separating biochemical mixture based on a highly specific interaction between antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid.
Affinity chromatography by Shiv kalia ( m.pharma analytical chemistry)Shiv Kalia
Detailed introduction of (Chromatography and Affinity Chromatography) and its theory, principle ,working ,application and limitation of Affinity Chromatography . This chromatography technique is also useful for GPAT ,UGC NET , GATE, DBT aspirants.
It includes basic knowledge about affinity chromatography along with its procedure and application. and brief description of various type of affinity chromatography.
Affinity chromatography is a sample purification technique,used primarily for biological molecules such as proteins.
1.Principle
2.Theory
3.Instrumentation
4. Applications
Chromatographic technique used for determining the biological activity of substances & to separate them from denatured or functionally different molecules. This type of chromatography is used to isolate enzymes & other proteins.
Introduction
Chromatography terms
History
Protein purification
Purpose
Chromatographic methods – a) Size exclusion
b) Ion exchange
c) Affinity
d) HPLC
Conclusion
Reference
In this presentation Steve will share with you how we transformed the Rally platform from a monolithic, three-tier SaaS architecture to a continuously deployed SOA system, piloting DevOps and Agile practices, while mapping and charting progress with Rally products and agile expertise.
For more information, please visit http://cainc.to/Nv2VOe
Affinity chromatography is a sample purification technique,used primarily for biological molecules such as proteins.
1.Principle
2.Theory
3.Instrumentation
4. Applications
Chromatographic technique used for determining the biological activity of substances & to separate them from denatured or functionally different molecules. This type of chromatography is used to isolate enzymes & other proteins.
Introduction
Chromatography terms
History
Protein purification
Purpose
Chromatographic methods – a) Size exclusion
b) Ion exchange
c) Affinity
d) HPLC
Conclusion
Reference
In this presentation Steve will share with you how we transformed the Rally platform from a monolithic, three-tier SaaS architecture to a continuously deployed SOA system, piloting DevOps and Agile practices, while mapping and charting progress with Rally products and agile expertise.
For more information, please visit http://cainc.to/Nv2VOe
Display technology is on demand with so many devices in the market. With constant operation of the device, the battery duration is limited. When organic solar cells are integrated with the display (organic LED), it serves as a dual purpose - decreasing light reflection (which otherwise would degrade contrast) and energy recycling (making using of the excess photons emitted from the display).
Separation is brought about through molecular sieving technique, based on the molecular size of the substances. Gel material acts as a "molecular sieve”.
Gel is a colloid in a solid form (99% is water).
It is important that the support media is electrically neutral.
Different types of gels which can be used are; Agar and Agarose gel, Starch, Sephadex, Polyacrylamide gels.
Computational modelling of drug disposition lalitajoshi9
computational modelling of drug disposition is the integral part of computer aided drug design. different kinds of tools being used in the prediction of drug disposition in human body. This topic in the CADD explains the details about the drug disposition, active transporters and tools.
Stability Indicating HPLC Method Development A Reviewijtsrd
High performance liquid chromatography is most powerful tools in analytical chemistry which assessing drug product stability. It is most accurate method for determining the qualitative and quantitative analysis of drug product. Forced degradation plays an important role in development of stability indicating analytical methodology. Stability indicating HPLC methods are used to separate various drug related impurities that are formed during the synthesis or manufacture of drug product. This article discusses the strategies and issues regarding the development of stability indicating HPLC system for drug substance. Forced degradation studies establish degradation pathways of drug substances and drug products. Forced degradation elucidate the possible degradation pathway of the drug substance or the active pharmaceutical ingredient in the drug product. At every stage of drug development practical recommendations are provided which will help to avoid failure. Rushikesh S Mulay | Rishikesh S Bachhav "Stability Indicating HPLC Method Development - A Review" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-5 | Issue-6 , October 2021, URL: https://www.ijtsrd.com/papers/ijtsrd46342.pdf Paper URL : https://www.ijtsrd.com/pharmacy/analytical-chemistry/46342/stability-indicating-hplc-method-development--a-review/rushikesh-s-mulay
Metabolomics is often described as the study of “the complete set of low molecular weight intermediates, which are context dependent, varying according to the physiology, developmental or pathological state of the cell, tissue, organ or organism”. In fact, metabolomics is a new term for an old science in which classical biochemical concepts are investigated. New and unique to the current research that is being conducted is the combination with genomics information and full system biology. In this refocus we will discuss the challenges in today's metabolomics research and how to address them
Computational modeling of drug dispositionPV. Viji
Computational modeling of drug disposition , Modeling techniques , Drug absorption , solubility , intestinal permeation , Drug distribution , Drug excretion , Active Transport , P-gp , BCRP , Nucleoside transporters , hPEPT1 , ASBT , OCT , OATP , BBB-choline transporter
COMPUTATIONAL MODELING OF DRUG DISPOSITION.pptxPoojaArya34
Computational modelling of drug disposition is the integral part of computer aided drug design. different kinds of tools being used in the prediction of drug disposition in human body. This topic in the CADD explains the details about the drug disposition, active transporters and tools.
Historically, drug discovery has focused almost exclusively on efficacy and selectivity against the biological target.
As a result, nearly half of drug candidates fail at phase II and phase III clinical trials because of undesirable drug pharmacokinetics properties, including absorption, distribution, metabolism, excretion, and toxicity (ADMET).
The pressure to control the escalating cost of new drug development has changed the paradigm since the mid-1990s. To reduce the attrition rate at more expensive later stages, in vitroevaluation of ADMET properties in the early phase of drug discovery has been widely adopted.Many high-throughput in vitro ADMET property screening assays have been developed and applied successfully .
For example, Caco-2 and MDCK cell monolayers are widely used to simulate membrane permeability as an in vitro estimation of in vivo absorption.
These in vitro results have enabled the training of in silico models, which could be applied to predict the ADMET properties of compounds even before they are synthesized.
1. Can hydrogel contribute to metabolomics?
A.M. Moschovi1,3, S.N. Yannopoulos2, V. Dracopoulos3,4, M. Klapa 1
forth|iceht
1 Metabolic Engineering & Systems Biology Lab,
2 Advanced Amorphous Materials and Nanomaterials Lab,
3 Ionic Liquids & Molten Salts Lab, 4 Microscopy & X-ray Diffraction Lab
Foundation for Research & Technology-Hellas, Institute of Chemical Engineering Sciences (FORTH/ICE-HT), Patras, GREECE
References :
1. Bolisay et al. Biomaterials 27 (2006) 4165–4168
2. P. Parmpi et al., Biomaterials 25 , 2004, 1969–1973
Acknowledgments:
Metabolomics is the high-throughput bioanalytical platform for the simultaneous
quantification of the relative concentration of free small metabolites in biological
systems. Metabolomics is presently at its standardization phase, during which data
acquisition and analysis protocols need to be optimized regarding performance and
resolution.
Introduction
Gas Chromatography(GC)-Mass Spectrometry(MS)
metabolomics will remain an integral component of
the metabolomics laboratory, due to distinct
advantages over the other utilized techniques.
However, a significant issue of GC-MS metabolomics is that the majority of measured
metabolite peaks have yet to be identified. Moreover, even identifiable peaks
corresponding to molecules of similar chemical structure, thus of similar GC retention
times and MS fragmentation patterns, cannot be easily differentiated. The phenomenon
affects the quantification of sugars (e.g. glucose, galactose) and their pyranoses, sugar-
alcohols and sugar-phosphates, accurate quantification of which is of importance in
many applications, as key intermediates of primary metabolism. Finally, the significantly
higher concentration of a metabolite (most commonly glucose) could affect the
derivatization and quantification of other molecules, thus its selective filtering is
desirable.
Hydrogels are insoluble crosslinked polymer network structures composed of hydrophilic
co- or homo-polymers which exhibit the ability to absorb significant amount of water.
Molecular imprinting (MI) in hydrogels is a technique in which functional groups of the
hydrogel are allowed to form a network around a template molecule. After the removal of
the template molecule, cavities with specific recognition sites and size are generated for
the preferential binding of the target over similar molecules.1 These materials are
candidates for molecular recognition, drug delivery, highly specific catalysis, quantitative
analysis and nanofiltration in conjunction with chromatographic techniques. We propose
to combine the recognition capabilities of MI polymer hydrogels with GC-MS
metabolomics to increase the resolution of the metabolic profiles. Initially, we will use a
MI hydrogel based on a crosslinked poly(allylamine) PAA∙HCl polymer with and/or
without D-glucose-6-phosphate imprinting, which has been reported as selective for
glucose over fructose1. Preliminary results on gelation dynamics and structure for the
bulk polymer have indicated their potential contribution to the monosaccharide
separation capabilities of the MI hydrogel.
Molecular Imprinting
Hydrogels are insoluble crosslinked polymer network
structures composed of hydrophilic polymers which
exhibit the ability to absorb significant amount of water.
Molecular imprinting
involves the formation of a complex between a
functional monomer and a template molecule
(poly-sacharides, viruses, proteins )1 with specific
chemical structure and functionality
(shape/ size/ functional groups).
Trends in Biotechnology, December 2010, vol 28, No12
After the template is
removed, the
product is a hetero-
polymer matrix with
specific recognition
capacities.
Monomer NaOH Crosslinker Template
200 100 26 3
Glucose specific HG
The binding capacity and
selectivity of molecular
imprinted Poly(allylamine)
hydrochloride PAA·HCl
polymer with the following
stoichiometry has been tested
in glucose and fructose
solutions.2,3
The presence of the template
during the synthesis procedure
resulted in the formation of
cavities with specific properties
.
The PAA·HCl pore before and after removing the template.
%
template
Glucose
binding(g/g)
Fructose
binding(g/g)
GPS-Na
0.50 0.58±0.02 0
1.00 0.54±0.03 0
1.5 0.50±0.01 0
No imprint 0.20±0.01 0
GPS-Ba 1.5 0,593±0.003 0.110±0.019
No
template
0.139±0.015 0.105±0.003
GPS-Ba 1.5 0.601±0.032 0.84±0.02
No
template
0.132±0.02 0.079±0.011
Non imprinted hydrogels exhibited
glucose selectivity and binding capacity
lower than the imprinted ones.
ConceptSome biological samples such as blood serum
and leaf extracts need special treatment due to
poor resolution in accurate quantification of
molecules having similar chemical structure and
the high concentration of some metabolites
(mainly glucose) which affects the derivatization
and quantification of other metabolites.
20 22 24 26 28
0
300000
600000
Intenisty
(counts)
t (min)
The latter problem can be
bypassed by selectively
filtrating the sample from the
metabolite in excess, before
the derivatization process in a
pre-column step, or by
separating metabolites with
similar structure using an
imprinted hydrogel prepared
with the target molecule as
the template.
Imprinted
Non- imprinted
Different kind of molecules(D-fructose, D-glucose, L-
glucose and D-gluconamide) have been tested as targets
for glucose-imprinted PAA·HCl hydrogel compared to the
non-imprinted polymer binding capabilities.4
It has been shown that the binding capacity of hydrogel was
not significantly enhanced by the imprinting.
Template D-Fructose D-glucose L-glucose D-gluconamide
none 0.308±0.019 0.649±0.038 0.548±0.045 0.795±0.026
Thus, we need to examine the role of the bulk in the binding capacities of the imprinted hydrogel for different targets
and investigate the reproducibility of the hydrogel synthesis process and binding capabilities for specific targets within
complex biological samples.
Previous Work
Gel synthesis
As prepared
SEM
XRD
DLS
0 20 40 60 80 100 120
3,5
4,0
4,5
5,0
5,5
6,0
6,5
7,0
7,5
RH
time(min)
10
-3
10
-2
10
-1
10
0
10
1
10
2
10
3
10
4
0,0
0,3
0,6
0,9
g
(2)
(t)-1
time (msec)
1min
18min
45min
65min
71min
74min
76min
90min
120min
10
-3
10
-2
10
-1
10
0
10
1
10
2
10
3
10
4
10
5
76min
74min
a.u.
t(msec)
120min
90min
71min
65min
45min
18min
1min
At the beginning of the
synthesis, three different
processes take place,
attributed to the monomers.
During the gelation at 60-
70min, a new process
appears corresponding to
aggregates and clusters
formation.
Later on the gelation, a new
process appears attributed
to the network formed in the
structure.
According to FTIR- ATR
spectra vibrational changes
are observed at 80-90min,
after the crosslinker was
added.
Conclusions
I. Gelation begins after 60-65 minutes at static
conditions at 25oC; process is highly reproducible.
II. NaCl crystals are formed from the neutralization
of the HCl groups of the monomer with the
NaOH.
III. After washing, NaCl crystals are totally removed
and pores are formed in the bulk of the HG.
IV. After the washing treatment, a small amount of
the template GPS-Ba is trapped in the porosity of
the HG.
V. Non imprinted HG are expected to exhibit binding
capabilities for small metabolites due to the
porosity formed by the NaCl crystals.
Future work
• Sugar binding capacity and selectivity of
imprinted and non-imprinted hydrogels will be
studied in standard composition & complex
biological mixtures (eg blood serum, leaf extracts)
• Physicochemical properties, structure and binding
capacities of imprinted hydrogels will be studied
with Dynamic Light Scattering, Infrared
Spectroscopy, GC-MS profiling.
Dynamic Light Scattering
Infrared Spectroscopy
Binding test
The binding properties of the bulk of the hydrogel were
investigated with glucose, fructose and ribitol aqueous solutions
and their mixtures.
The concentration of the binding solution was 50mg/ml.
Samples were pepared for the GC-MS measurements according
to Ref. 5
•Dissolution of the polymer in H2O (25% w/v)
•Addition of the template
•Neutralization of the amine groups with NaOH
•Addition of the crosslinker EPI
•Gelation
•Washing with NaOH to remove unreacted reagents
•Removal of the NaOH excess with H2O
•Dry at 50oC
No selectivity was observed for the template-free hydrogel
3. Wizeman W.J. et al. Biomaterials 22, 2001, 1485-1491
4. Fazal et al. Bioorganic & Medicinal Chemistry Letters 17, 2007, 235-238
5. Kanani et al. J of Chromatography B, 871(2008) 191-201 National Strategic Reference Framework
FUNDING:
Template GPS-Ba washed(NaOH/H2O)Template free /washed (H2O) Template free /washed(NaOH/H2O)
Template free
As prepared Dried
Imprinted
As prepared Washed
NaCl crystals and template
molecules are removed after
washing treatment for imprinted
and template free hydrogels.
200nm
200nm
2μm10μm20μm
2μm
100μm
2μm
Template and NaCl
removal enable the
formation of porosity
in the bulk