Column Chromatography

 Column Chromatography | MIT Digital Lab Techniques Manual

By MIT Opencourse

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Column Chromatography

By Lisa Nichols

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Colorless Column

By Lisa Nichols

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How to write Laboratory Note Book

By Dr. Arnab Samanta

Introduction

Experiments by the scientists in the laboratory lead to scientific inventions. The time spent in laboratory work is mainly devoted to experimental, theoretical and analytical work. But, unless properly recorded for future use, the experimental work could become a futile exercise.

What is a laboratory note book?

Laboratory note book is a basic tool for recording details of any experimental work.

Purpose of maintaining a laboratory note book

1. A detailed laboratory note book is the best and most direct evidence of the work performed.
2. Intellectual property rights protection (IPR): The laboratory notebook is a base document with which a scientist’s IPR may be protected. Exact details, date of conception, experimental observations and results, and chronological record of a work are essential parts of evidence for patent applications.
3. Scientific repeatability: Based on the content of a laboratory notebook another scientist should be able to repeat the work performed, using only the notebook as a guide.
4. Project accountability: Often a project is run with the help of some funding body. To assure the funding body that their investments are being effectively and wisely utilized, accurate, complete, authentic and reliable records of research data is essential. This proves the scientific integrity of the staff as well as the institute.

N.B. In December 1994, the US Government passed a bill on the Agreement on Trade Related Aspects of Intellectual property Rights (TRIPS) of the General Agreements on Tariff and Trade (GATT). This bill allows foreign countries, including India, to apply for US Patent. To take the advantage of the US Patent Laws the Government of India has established guidelines for maintenance of laboratory note books. All the laboratories in India, including the CSIR Laboratories follow these general guidelines.

Maintaining the Laboratory Note Book

1. The note book should be bound, so that pages cannot be removed and replaced.
2. The pages should be numbered consecutively, preferably by a printer or a counter.
3. The pages may be plain, bordered, lined or with grid.
4. The first page of the notebook should list the principal investigator, the names of the other investigators, the title of the study and the date of beginning of the study. The aim of the study and the problems the study attempts to address may also be indicated.
5. If more than one notebook is used for a project, they should be serially numbered.
6. All entries should be in non-erasable ink.
7. “White outs” are not allowed. Erasures are not allowed. In case of an error the entry should be crossed out; taking care to make sure that the entry was legible (i.e. readable).
8. Date written only in numerals (e.g. 29/02/2008) may cause confusion, hence it better to follow the format 29 Feb 2008. Proof of the date is crucial especially during patenting of an invention or when a dispute arises between two inventors (that who should be called the ‘first inventor’). Under US Patent Laws the ‘first to invent’ is granted the patent for an invention rather than the ‘first to file’.
9. If a blank page is not used then it must be voided by drawing a corner-to-corner line. Every bit of every page must be filled and legible either with information or with a mark that voides a section.
10. The language used in a note book should be objective and factual. The past tense (e.g. ‘was heated’) should be used to describe experiments that were actually performed.
11. When recording an experiment, it is important to include enough information, so that the experiment will be reproducible. These include information relating to the equipment used, time, condition and method of experiment.
12. It is advisable to record all the entries in the table of content (TOC) as one goes along. It is preferable to include multiple levels in a TOC to include subheadings of an experiment or part of it. The TOC may take two or more pages at the beginning of the lab notebook.
13. All abbreviations and acronyms must be defined the first time they appear in the lab notebook.
14. It is important to include conceptions (ideas) and not just data in the notebook. An idea concerning, what the invention is, what problem it addresses, and how it is different from what was done earlier, should be entered in the laboratory notebook. Comments should be made objective and accurate. Derogatory comments about the work of others should be avoided. It is often useful to record what occurred at laboratory group meetings, especially suggestions regarding the project work or experiment. This is helpful in documenting conception and inventorship.
15. Entries made by any person other than the person to whom the notebook belongs must be dated and signed by the person making the entry. Data that is generated may be incorporated in the notebook by photocopying (e.g. graphs) and stapling into the notebook. Data that cannot be stapled into the notebook should be maintained in an orderly manner and its location should be cross-referenced in the notebook.
16. An independent witness, i.e. some one who understands the technology but will not be named as a co-inventor of the invention, should sign and date each entry after a statement like: “Read and understood by …”. The witness can sign the entries on daily, weekly or monthly basis. No changes should be made to an entry after witnessing of a page. If changes is made, the witness should re-witness the entry by signing and dating next to the correction.

Archiving and Categorization

Notebooks are permanent records of a research project, and are stored securely for years and sometimes for centuries. Laboratory notebooks that relate to inventions on which patents are granted must be preserved for the life of the patent plus six years.

Laboratory notebooks are categorized by levels of use as follows:

1. A working laboratory notebook – one that is in current use.
2. An active laboratory notebook – one that is complete but is required for reference.
3. An inactive laboratory notebook – one that is complete and currently not required for quick reference.

·         Most institutes permit the investigators to make copies of the primary data in the laboratory notebook for his or her own use, but laboratory notebooks are not permitted to be removed from the laboratory.

·         Lab notebooks of eminent scientists and Nobel Laureates are preserved carefully and studied for centuries.

Laboratory notebooks of the future

The increasing use of computers in research indicates that very soon we may use electronic laboratory notebooks (e-laboratory notebook). As software is increasingly being used in research, the number of computer-assisted discovery programs will also increase, making the use of e-lab notebook inevitable.

Conclusion

• Scientists have a responsibility to science. Confidence in reproducibility of the results is fundamental to another scientist who would like to build upon the earlier results of another scientist. Science is built on trust and the trust is most inspired when the original data exists for review.
• The laboratory note books are not clean documents as we are taught from our childhood. It is evident from the lab note books of the famous scientists.

Examples of Note Books of Famous Scientists

Thomas Alva Edison (T.A. Edison)

Fig. Thomas Alva Edison [1847-1931]

The most prolific inventor of American History was Thomas Alva Edison. He had 1093 patents under his authorship. He was a very disciplined scientist. He maintained all his works in his Lab Note books.

Fig. A bound Lab note book of T. A. Edison

Fig. A TO DO LIST from the lab note book of Thomas Alva Edision

Sir Isaac Newton

Fig. A page from the note book of Newton

Sir Alfred Nobel

Fig. Two pages from the Lab note book of Sir Alfred Nobel

Alexander Graham Bell - the inventor of telephone

Fig. Two pages from the lab notebook of Alexander Graham Bell

Preparation of Liposomes-I

 Liposomes Introduction and Preparation by Zoraiz Haider

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Lipids for Liposomes: Selection, Preparation and Application

Di Bush, Avanti Polar Lipids

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Microfluidic Production of Liposomes

Yvonne Perrie, Strathclyde University

• Microfluidic production of liposome
• Visual appearance of liposome under microscope, particile size distribution, 
• Phase transition temperature

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Plasma membrane | Transition temperature

By Shantanu Kolhe

• Behavior of lipids in the fluidity of plasma membrane.

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Cell membrane fluidity and role of cholesterol in membrane fluidity

By Shomu's Biology

• Cholesterol has a big bulky hydrophobic body and a very small hydrophilic group (-OH),
• In this video the role of Cholesterol in maintaining the membrane fluidity is discussed.
• At higher temperature, cholesterol decreases membrane-fluidity. Cholesterol provides large surface area for hydrophobic interaction with the long tails of the surrounding lipid chains of the phospholipids. Thus makes the lipid membrane more rigid.
• At lower temperature, cholesterol molecules push the closely packed long tails of the phospholipids. Thus cholesterol increases the membrane-fluidity.
• Therefore, Cholesterol molecules provide the optimum fluidity of the lipid bilayers at both higher and lower temperatures.

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Lab 5 Synthesis and Characterization of Liposomes

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Making Liposomes

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HOW TO MAKE LIPID NANOPARTICLES - A day in the life of a PhD

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Making Liposomal Vitamin C: a tutorial
by Henrik Eiriksson

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Particle size analysis of suspension

Determination of particle size of suspension by Pipette Method

Determination of particle size of suspension by Hydrometer Method

Particle size analysis

Sieve nalysis

Vaccine Adjuvants

Freund's adjuvant

Jules T Freund

It is an w/o emulsion made from (i) aqueous phase - a solution of antigen, (ii) oil-phase - mineral oil and (iii) an emulsifier - Arlacel A (Dianhydro-D-mannitol monooleate). When this emulsion was injected in animal it was found to act as an immunopotentiator or booster to immunity reaction.

Complete Freund's Adjuvant (CFA or FCA)

It is composed of inactivated and dried mycobacteria (usually M. tuberculosis) which acts as the antigen. The antigen is administered in the form of w/o emulsion.

• CFA stimulates the cell-mediated immunity. It potentiates the T-helper cells that lead to the production of certain immunoglobulins and effector T-cells.
• The CFA is very toxic to animals and human beings as well. It produces painful reaction and causes tissue damages (necrosis). 
• FCA should be administered subcutaneously or intraperitoneally because intradermal injections may cause skin ulceration and necrosis, intramuscular injection may lead to permanent muscle lesion and intravenous injections may produce lipid embolism.

Incomplete Freund's Adjuvant

The w/o emulsion is devoid of any antigen, i.e. it is the emulsion only.

How do vaccines work

Histological toxicity study of subcutaneous in situ gel

Materials

Male Wistar rats, normal saline solution, gel formulation, hematoxylin-eosin stain, microtome, paraffin, compound microscope.

Procedure

• Twenty four male Wistar rats weighing between 190-225g are used as test animals. They are divided into two groups.

Group-I:Control Gr:Physiological saline 0.3ml

Group-II:Test Gr:In situ gel formulation 0.3ml

• The formulations are given subcutaneously. The rats are sacrificed at 1st, 7th and 14th day after the begining of the study.
• The subcutaneous tissue at the injection site is separated and fixed by 10% formalin solution for three days. The fixed subcutaneous tissue is embedded in paraffin and sliced into 3micron thin slices.
• To evaluate the inflammatory reaction at the site of injection, the comparison of the numbers of neutrophils, eosinophils and macrophages between Group-I and Group-II is performed. Neutrophils and eosinophils are counted in hematoxylin-eosin stained sections. Macrophages are counted in immunohistolchemical staining sections.

References 

Xin, C., Lihong, W., Qiuyuan, L., Hongzhuo, L., 2014. Injectable long-term control-released in situ gels of hydrochloric thiothixene for the treatment of schizophrenia: Preparation, in vitro and in vivo evaluation. International Journal of Pharmaceutics 469, 23–30.

Dissolution study of polylactic acid based in situ gels

Dissolution study of polylactic acid based in situ gels [prolonged release for 12 days]

In situ gel delivery systems become viscous when the solution is immersed in dissolution medium (or body fluid) at body temperature. The gel may take very long time to get completely dissolved, thus slowly releasing the drug.

Materials

USP Dissolution rate test apparatus, graduated pipette (2ml cap.), phosphate buffer solution (pH 7.4), sodium azide, 

Procedure

• Gel formulation of 10 ml is taken in a small porcelain or glass cup and placed at the bottom of  a USP-2 dissolution rate test apparatus (paddle type). The round bottom jar is filled with 900ml phosphate buffer (pH7.4). Temperature is maintained at 37⁰C. 
• For a prolong released product the time of dissolution may require 12 days or more. In such case, to prevent bacterial growth, sodium azide (final concentration in the 900 ml dissolution medium 0.02%w/v  ) is used.
• The paddle speed is fixed at 50 rpm. Sample (1ml) is collected from the dissolution medium at 1, 3, 6, 9, 12 days interval. After each sampling the round bottom beaker is replenished with 1ml of fresh dissolution medium pre-warmed at 37⁰C.
• The samples are filtered by centrifugation and finally analyzed with HPLC.

References

Xin, C., Lihong, W., Qiuyuan, L., Hongzhuo, L., 2014. Injectable long-term control-released in situ gels of hydrochloric thiothixene for the treatment of schizophrenia: Preparation, in vitro and in vivo evaluation. International Journal of Pharmaceutics 469, 23–30.