Simultaneous equation method for uv spectroscopy

SIMULTANEOUS EQUATION METHOD OR VIERODT'S METHOD

If  a  sample  contains  two  absorbing  drugs  (X  and  Y)  each  of  which  absorbs  at  the  λmax  different  from  the  other,  it  may  be  possible  to  determine  both  drugs  by  the  technique  of  simultaneous  equations (Vierodt's  method),  provided  certain  criterias  apply.  The information required is (a) The aborptivities of X at and λ1 and λ2 are ax1 and ax2 respectively (b) The aborptivities of Y at and λ1 and λ2 are ay1 and ay2  respectively.  (c) The absorbances of  the diluted sample  at  λ1  and  λ2  are A1  and  A2  respectively.  Let  Cx  and  Cy  be  the  concentrations  of  X  and  Y  respectively  in  the  diluted  sample.  Two equations are constructed based upon the  fact that at λ1 and λ2, the  absorbance of the mixture is the sum of the individual absorbance of X and Y.

 At λ1             A1 = a X1 b Cx + aY1b Cy ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ (1)        

 At λ2            A2 = a X2 b Cx + aY2 b Cy‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ (2) 

   For measurements in 1 cm cells b=1           

   Rearrange eq. (2) 

  A2  ‐ a X2 b Cx            Cy =   ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐                                                            aY2 

Grandmother's smoking in pregnancy

Summary:

Scientists have looked at all 14,500 participants in Children of the 90s and found that if a girl's maternal grandmother smoked during pregnancy, the girl is 67 percent more likely to display certain traits linked to autism, such as poor social communication skills and repetitive behaviors.

Scientists from the University of Bristol have looked at all 14,500 participants in Children of the 90s and found that if a girl's maternal grandmother smoked during pregnancy, the girl is 67% more likely to display certain traits linked to autism, such as poor social communication skills and repetitive behaviours.

The team also found that if the maternal grandmother smoked, this increased by 53% the risk of her grandchildren having a diagnosed autism spectrum disorder (ASD).

These discoveries suggest that if a female is exposed to cigarette smoke while she is still in the womb, it could affect the developing eggs -- causing changes that may eventually affect the development of her own children. Further research is now needed to find out what these molecular changes might be, and to see whether the same associations are present in other groups of people.

Unlike the analysis of autistic traits, which was based on over 7,000 participants, the 177 diagnosed with ASD were too few to analyse grandsons and granddaughters separately.

The discovery, published today in Scientific Reports, is part of an ongoing, long-term study of the effects of maternal and paternal grandmother's smoking in pregnancy on the development of their grandchildren, who are all part of Children of the 90s. By using detailed information collected over many years on multiple factors that may affect children's health and development, the researchers were able to rule out other potential explanations for their results.

The incidence of ASD has increased in recent years, and while some of this increase is undoubtedly down to improved diagnosis, changes in environment or lifestyle are also likely to play a role. The researchers also stress that many different factors, including genetic variation, are believed to affect an individual's chances of developing ASD.

Past studies of maternal smoking in pregnancy and ASD in children have been inconclusive. Going back a generation has revealed an intergenerational effect, which interestingly is most clear cut when the mother herself did not smoke in pregnancy.

The reasons for this are not entirely clear but Professor Marcus Pembrey, one of the paper's authors, says: 'In terms of mechanisms, there are two broad possibilities. There is DNA damage that is transmitted to the grandchildren or there is some adaptive response to the smoking that leaves the grandchild more vulnerable to ASD. We have no explanation for the sex difference, although we have previously found that grand-maternal smoking is associated with different growth patterns in grandsons and granddaughters.

'More specifically, we know smoking can damage the DNA of mitochondria -- the numerous "power-packs" contained in every cell, and mitochondria are only transmitted to the next generation via the mother's egg. The initial mitochondrial DNA mutations often have no overt effect in the mother herself, but the impact can increase when transmitted to her own children.'

Professor Jean Golding, another author, added: 'We already know that protecting a baby from tobacco smoke is one of the best things a woman can do to give her child a healthy start in life. Now we've found that not smoking during pregnancy could also give their future grandchildren a better start too. We have started studying the next generation of participants (COCO90s), so eventually we will be able to see if the effect carries down from the great-grandparents to their great-grandchildren too.'

Dr Dheeraj Rai, another author, added: 'We still do not know why many children develop autism and behaviours linked to it. The associations we observe raise intriguing issues on possible transgenerational influences in autism. Future research will help understand the meaning and mechanisms behind these findings. The National Autistic Society website contains a wealth of information about autism and details on how and where to seek advice.'

Alycia Halladay, PhD, chief science officer at the Autism Science Foundation (USA), said: 'To date, research into the causes of autism has been limited to studying maternal or paternal exposures during pregnancy. By utilizing a birth cohort in the United Kingdom [Children of the 90s], scientists are able to go back a generation to examine the role of grandparental exposures, presumably through germ line mutations and epigenetic modifications. Hopefully, grandparental exposures will continue to be investigated to better understand this mechanism.'

Source: https://www.sciencedaily.com/releases/2017/04/170427091740.htm

Ability of stem cells expanded to regrow any tissue type

Source:

Salk Institute

Summary:

A new technique, which allows scientists to generate both embryonic and non-embryonic tissues from cultured stem cells, is a step toward growing donor organs and replacement tissues to combat aging and diseases.

Human EPS cells (green) can be detected in both the embryonic part (left) and extra-embryonic parts (placenta and yolk sac, right) of a mouse embryo.

Credit: Salk Institute

When scientists talk about laboratory stem cells being totipotent or pluripotent, they mean that the cells have the potential, like an embryo, to develop into any type of tissue in the body. What totipotent stem cells can do that pluripotent ones can't do, however, is develop into tissues that support the embryo, like the placenta. These are called extra-embryonic tissues, and are vital in development and healthy growth.

Now, scientists at the Salk Institute, in collaboration with researchers from Peking University, in China, are reporting their discovery of a chemical cocktail that enables cultured mouse and human stem cells to do just that: generate both embryonic and extra-embryonic tissues. Their technique, described in the journal Cell on April 6, 2017, could yield new insights into mammalian development that lead to better disease modeling, drug discovery and even tissue regeneration. This new technique is expected to be particularly useful for modeling early developmental processes and diseases affecting embryo implantation and placental function, possibly paving the way for improved in vitro fertilization techniques.

"During embryonic development, both the fertilized egg and its initial cells are considered totipotent, as they can give rise to all embryonic and extra-embryonic lineages. However, the capture of stem cells with such developmental potential in vitro has been a major challenge in stem cell biology," says Salk Professor Juan Carlos Izpisua Bemonte, co-senior author of the paper and holder of Salk's Roger Guillemin Chair. "This is the first study reporting the derivation of a stable stem cell type that shows totipotent-like bi-developmental potential towards both embryonic and extra-embryonic lineages."

Once a mammalian egg is fertilized and begins dividing, the new cells segregate into two groups: those that will develop into the embryo and those that will develop into supportive tissues like the placenta and amniotic sac. Because this division of labor happens relatively early, researchers often can't maintain cultured cell lines stably until cells have already passed the point where they could still become either type. The newly discovered cocktail gives stem cells the ability to stably become either type, leading the Salk team to dub them extended pluripotent stem (EPS) cells.

"The discovery of EPS cells provides a potential opportunity for developing a universal method to establish stem cells that have extended developmental potency in mammals," says Jun Wu, a senior scientist at Salk and one of the paper's first authors. "Importantly, the superior interspecies chimeric competency of EPS cells makes them especially valuable for studying development, evolution and human organ generation using a host animal species."

To develop their cocktail, the Salk team, together with the team from Peking University, first screened for chemical compounds that support pluripotency. They discovered that a simple combination of four chemicals and a growth factor could stabilize the human pluripotent stem cells at a developmentally less mature state, thereby allowing them to more efficiently contribute to chimera (a mix of cells from two different species) formation in a developing mouse embryo. They also applied the same factors to mouse cells and found, surprisingly, that the newly derived mouse stem cells could not only give rise to embryonic tissue types but also differentiate into cells from the extra-embryonic lineages. Moreover, the team found that the new mouse stem cells have a superior ability to form chimeras and a single cell could give rise to an entire adult mouse, which is unprecedented in the field, according to the team.

"The superior chimeric competency of both human and mouse EPS cells is advantageous in applications such as the generation of transgenic animal models and the production of replacement organs," adds Wu. "We are now testing to see whether human EPS cells are more efficient in chimeric contribution to pigs, whose organ size and physiology are closer to humans." Human EPS cells, combined with the interspecies blastocyst complementation platform as reported by the same Salk team in Cell in January 2017, hold great potential for the generation of human organs in pigs to meet the rising demand for donor organs.

"We believe that the derivation of a stable stem cell line with totipotent-like features will have a broad and resounding impact on the stem cell field," says Izpisua Belmonte.

AKCENT GROUP "Charak Institute of pharmacy, Mandleshwer

AKCENT GROUP 

Charak Institute of Pharmacy, Mandleshwer Dist. Khargone (M.P.)

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Charak 8th Batch 

Our Facuilty Members

Solanki Sir. Patel Sir, Jaiswal sir ,Patidar sir .

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Charak 7th Batch Student

Jordan Sir, Sharma sir Patidar Sir

My First Poster Presentation 2013

THE PHARMACY ACT, 1948

INTRODUCTION

In India there was no restriction to practise the profession of pharmacy. One could practise this profession as any other profession. Persons, having no knowledge and having no education in pharmacy or pharmaceutical chemistry or pharmacology, were engaged in this profession. Hundreds of cases were brought to the notice of the Government wherein the compounding, mixing, or dispensing of medicines was being done by persons who were not adequately educated in this line. The system was causing great harm to the health of people by wrong compounding, mixing or dispensing. It was found necessary to enact a law for the regulation of the profession and practice of pharmacy. To achieve this goal the Pharmacy Bill, 1947 was introduced in the Legislature which was later referred to the Select Committee. The recommendations of the Selection Committee were incorporated in the Bill.

STATEMENT OF OBJECTS AND REASONS

It is desirable that, as in most other countries, only persons who have attained a minimum standard of professional education should be permitted to practise the Profession of Pharmacy. It is accordingly proposed to establish a Central Council of Pharmacy, which will prescribe the minimum standards of education and approve courses of study and examinations for Pharmacists, and Provincial Pharmacy Councils, which will be responsible for the maintenance of provincial registers of qualified pharmacists. It is further proposed to empower Provincial Governments to prohibit the dispensing of medicine on the prescription of a medical practitioner otherwise than by, or under the direct and personal supervision of, a registered pharmacist.

ACT 8 OF 1948

The Pharmacy Bill, 1947, having been passed by the Legislature received its assent on 4th March, 1948. It came on the Statute Book as THE PHARMACY ACT, 1948 (8 of 1948).

LIST OF AMENDING ACTS AND ADAPTATION ORDERS

1. The Adaptation of Laws Order, 1950.

2. The Adaptation of Laws (No.3) Order, 1956.

3. The Pharmacy (Amendment) Act, 1959 (24 of 1959).

4. The Pharmacy (Amendment) Act, 1976 (70 of 1976).

5. The Pharmacy (Amendment) Act, 1982 (22 of 1982).

6. The Delegated Legislation Provisions (Amendment) Act, 1985 (4 of 1986).

______________________

Packaging Technology

DEFINITION: 

Packing: Packing consists of enclosing an individual item, or several items, in a container, usually for shipment or delivery. This operation is mostly done by hand and machine. Pharmaceutical

Packaging: Pharmaceutical packaging means the combination of components necessary to contain, preserve, protect & deliver a safe, efficacious drug product, such that at any time point before expiration date of the drug product, a safe & efficacious dosage form is available.

Types of Packaging Systems: 

1.  Primary package system: Made up of those package components & subcomponents that come into direct contact with the product, or those that may have a direct effect on the product shelf life. 
2. Secondary or tertiary package system: Includes cartons, corrugated shippers & pallets

Packaging must meet the following Requirements: [ideal requirements] 

1.  Protect the preparation from environmental conditions. 
2. Non-reactive with the product and so does not alter the identity of the product 
3.  Does not impart tastes or odors to the product 
4.  Nontoxic 
5.  FDA approved 
6. Protect the dosage form from damage or breakage 
7. Meet tamper-resistance requirements, wherever applicable. 
8. Adaptable to commonly employed high-speed packaging equipments.

Criteria for the Selection of package type and package material: 

1. Stability 
2.  Compatibility with the contents 
3. Strength of container and the degree of protection required 
4.  Moisture-proofness 
5.  Resistance to corrosion by Acids or Alkalis 
6.  Resistance to grease 
7. Protection against salt 
8.  Resistance to microorganisms 
9.  Resistance to insects and rodents 
10. Resistance to differences in temperature 
11. Protection against light, fire and pilferage
12.  Odor retention and transmission 
13.  Aesthetic effect 
14. Cost 
15. Machine suitability of packaging and the filling method.

Possible Interactions between primary packaging materials and the included pharmaceutical product:

1. The release of chemicals from components of the packaging materials 
2. The release of visible and/or sub visible particles 
3.  The absorption or adsorption of pharmaceutical components by the packaging materials 
4.  Chemical reactions between pharmaceutical product & the packaging materials 
5. The degradation of packaging components in contact with the pharmaceutical products
6.  The influence of the manufacturing process (e.g. sterilization) on the container.

Packaging materials & closures: 

•  Glass 
• Plastic 
• Metals 
•  Paper and Board 
•  Rubber 
•  Cotton 
• Adhesives and Inks 
• Closures

GLASS CONTAINERS:

           Advantages:

1.  Superior protective qualities
2. Economical
3. Readily available in a wide variety of sizes & shapes 
4.  Essentially chemically inert, impermeable, strong and rigid 
5.  Has FDA clearance 6. Does not deteriorate with age
6. Provides an excellent barrier against every element except light with a proper closure system. Colored glass, especially amber, can give protection against light. 

             Disadvantages:

1.  Fragility 
2.  Heavy Weight.

         Composition of glass

• Mainly made up of

1. Sand – pure silica 
2.  Soda-ash – sodium carbonate 
3. Limestone – calcium carbonate 
4. Cullet – broken glass that is mixed with the batch & acts as a fusion agent for the entire mixture

The most common cations found in pharmaceutical glassware are silicone, aluminum, boron, sodium, potassium, calcium, magnesium, zinc & barium. The only anion of consequence is oxygen.

TYPES OF GLASS

 

Type I – Borosilicate Glass 

Type II – Treated Soda-Lime Glass

 Type III – Regular Soda-Lime Glass 

Type NP – General Purpose Soda-Lime Glass

Coming Soon...........

8 reasons why you may not trust Fizzle as far as you can throw us yet.

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1. “Isn’t just about everything you share available for free on the internet?”

Wow, starting out with a doozie. Ok, let’s talk about it.

Ideas about the foundations of business are available ALL OVER the web.

You can spend TONS of time clicking around the web learning bits and pieces.

The danger, of course, is that many people never move beyond that - spending A LOT of time clicking around.

Onesie-twosie learning will never be as curated, ordered, organized and action oriented as The Fizzle Roadmap.

We know the common pitfalls, we help you avoid them and make progress on your business every single week. This is what the Roadmap and community at Fizzle were designed for, and this is what our members gladly pay for each month.

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2. “Aren’t you too young to be business trainers?”

Some of us really do have baby faces :)

First, we have deep experience in both the new and old business worlds. Fortune 500 consultant, head of sales at a massive startup, highly sought-after designer… these are the careers we built before joining Fizzle.

Second, we were the team of choice when a few massive bloggers needed help with strategy and design. (That was back when we did client work.) They believed in how we look at new business. (Spoiler Alert: it paid off well for them.)

Third, some of our courses aren’t actually taught by us, they’re taught by leading experts. (Look for more of these in the future as we diversify the course library.)

But what speaks most for us is the success of our members. People like Adam, Bree, Val and Tom who joined Fizzle with an idea and are now earning a full-time living on their own terms.

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3. “Are you just out to make a quick buck?”

We priced Fizzle membership at an affordable $35/month (about a dollar a day), and included a free trial and an easy “click to cancel” button to stop being billed immediately.

If we wanted to make a quick buck we’d have a big price tag, lots of marketing and it’d be hard for you to get a refund.

No, we’re not out to make a quick buck. Instead, we designed ourselves into a business that only survives in a long-term relationship with you.

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4. “I’m worried your training will be too basic or too general.”

Yes, this is a very common question.

From idea to initial audience, from first product to audience growth, from creation to sales and marketing, there are actions.

Some actions require you to know certain concepts and ideas, so there’s some learning you may need there.

Maybe more than anything else, these actions, done in a particular order, are the most important ingredient in making your idea a reality and growing your business.

Fizzle will focus you on those actions regardless of how advanced or basic your understanding is at this point.

Besides that, the community and our weekly coaching calls go a long way to answering specific questions that might be unique to your business.

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5. “I have other business trainers I already trust.”

I totally understand.

I mean, we are very unique here at Fizzle. We have a very humane, open and fun way of looking at business and have designed ourselves into a business model that requires us to be excellent and honest.

And, obviously, the special community here is without equal in the world of online business.

Also, we’re very enthusiastic about dancing. It’s just something we are committed to (Steph in particular).

So, we have a unique way of looking at things and a very special community, and we just might be the hardest working business in business, but we’re not hurt if you like someone else more than us…

… well, I mean a little… but we’ll be OK.

(PS. if this is where you are, reply and tell us who you like so much. They must be doing a great job, and maybe we can collaborate.)

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6. “I doubt I’ll have time for training right now.”

This is a really good point. Thanks for your candor.

So, here’s what we’ve done at Fizzle to design for the realities of modern life.

We’ve broken up each course and each section of the roadmap into bite-sized chunks so you can do them at your own pace.

We also have a dashboard that makes it easy to pickup exactly where you left off.

And every friday we send out a short email called “This Week in Fizzle,” sharing popular conversations from the forums. (This is one of my favorite things we do.)

Managing time is a massive challenge for just about all small business builders. We’ve designed the training and our weekly “This Week in Fizzle” community email to fit Fizzle into your life, not the other way around.

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7. “I don’t think I’m ready to pay money for training.”

Wow, I really understand this one. Thank you for your honestly.

Now, let’s take that honesty a little further and realize that what you might be saying is this: I am not yet very serious about starting my own business.

First of all, please know that is a very OK thing to say, and brave. Many people think themselves very serious about business when they’re really just playing and pretending.

And second, maybe just beware of staying in that spectator position for too long… your future self might come to resent you. (Lord knows there’s things I wish I would have started sooner.)

So, can you still be interested in starting a business without paying for professional training? Yes. Fizzle’s blog and podcast, hopefully, are very inspiring places to keep learning.

And, when it come time to invest in yourself-if your goal is to earn a living doing something you care about as a freelancer, creative or entrepreneur-I hope you consider Fizzle membership. (At the very least it’ll be the most affordable excellent training you’ll likely find :)

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8. “It feels kinda weird that you’re so up front about this stuff.

I know, even as I write this I’m, like, “this isn’t normal!”

I hope it’s not too weird.

As I said, we can’t afford to be dishonest because people would just stop paying the $35/month for Fizzle.

We don’t want everyone to join Fizzle, but we really want the right kind of person to know about it. So, my bet is that brutal honesty will help attract awesome people.

VALIDATION OF ANALYTICAL PROCEDURES

Introduction 

This document presents a discussion of the characteristics for consideration during the validation of the analytical procedures included as part of registration applications submitted within the EC, Japan and USA. This document does not necessarily seek to cover the testing that may be required for registration in, or export to, other areas of the world. Furthermore, this text presentation serves as a collection of terms, and their definitions, and is not intended to provide direction on how to accomplish validation. These terms and definitions are meant to bridge the differences that often exist between various compendia and regulators of the EC, Japan and USA. 

The objective of validation of an analytical procedure is to demonstrate that it is suitable for its intended purpose. A tabular summation of the characteristics applicable to identification, control of impurities and assay procedures is included. Other analytical procedures may be considered in future additions to this document. 

Types of Analytical Procedures to be Validated The discussion of the validation of analytical procedures is directed to the four most common types of analytical procedures:

- Identification tests; - Quantitative tests for impurities' content.

 - Limit tests for the control of impurities,

 - Quantitative tests of the active moiety in samples of drug substance or drug product or other selected component(s) in the drug product.  

Although there are many other analytical procedures, such as dissolution testing for drug products or particle size determination for drug substance, these have not been addressed in the initial text on validation of analytical procedures. Validation of these additional analytical procedures is equally important to those listed herein and may be addressed in subsequent documents.

A brief description of the types of tests considered in this document is provided below. 

- Identification tests are intended to ensure the identity of an analyte in a sample. This is normally achieved by comparison of a property of the sample (e.g., spectrum, chromatographic behavior, chemical reactivity, etc) to that of a reference standard.

- Testing for impurities can be either a quantitative test or a limit test for the impurity in a sample. Either test is intended to accurately reflect the purity characteristics of the sample. Different validation characteristics are required for a quantitative test than for a limit test.

- Assay procedures are intended to measure the analyte present in a given sample. In the context of this document, the assay represents a quantitative measurement of the major component(s) in the drug substance. For the drug product, similar validation characteristics also apply when assaying for the active or other selected component(s). The same validation characteristics may also apply to assays associated with other analytical procedures (e.g., dissolution).

The objective of the analytical procedure should be clearly understood since this will govern the validation characteristics which need to be evaluated. Typical validation characteristics which should be considered are listed below: 

• Accuracy 
• Precision
•  Repeatability
•  Intermediate 
• Precision
•  Specificity 
• Detection Limit
•  Quantitation Limit 
• Linearity Range  

Each of these validation characteristics is defined in the attached Glossary. The table lists those validation characteristics regarded as the most important for the validation of different types of analytical procedures. This list should be considered typical for the analytical procedures cited but occasional exceptions should be dealt with on a case-by-case basis. It should be noted that robustness is not listed in the table but should be considered at an appropriate stage in the development of the analytical procedure.

Furthermore revalidation may be necessary in the following circumstances:

- changes in the synthesis of the drug substance.

- changes in the composition of the finished product.

 - changes in the analytical procedure.

- signifies that this characteristic is not normally evaluated

 + signifies that this characteristic is normally evaluated

1.   In cases where reproducibility  has been performed, intermediate precision is not needed 
2.  Lack of specificity of one analytical procedure could be compensated by other supporting analytical procedure(s)
3.   May be needed in some cases 

ANALYTICAL PROCEDURE 

The analytical procedure refers to the way of performing the analysis. It should describe in detail the steps necessary to perform each analytical test. This may include but is not limited to: the sample, the reference standard and the reagents preparations, use of the apparatus, generation of the calibration curve, use of the formulae for the calculation, etc. 

SPECIFICITY

 Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include impurities, degradants, matrix, etc. 

Lack of specificity of an individual analytical procedure may be compensated by other supporting analytical procedure(s).

This definition has the following implications: 

Identification:         To ensure the identity of an analyte. 

Purity Tests:          To ensure that all the analytical procedures performed allow an accurate statement of the                               content of impurities of an analyte, i.e. related substances test, heavy metals, residual                                     solvents content, etc.  

Assay (content or potency): 

                                          To provide an exact result which allows an accurate statement on the content or                                            potency of the analyte in a sample. 

ACCURACY

 The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. 

PRECISION 

The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility.

 Precision should be investigated using homogeneous, authentic samples. However, if it is not possible to obtain a homogeneous sample it may be investigated using artificially prepared samples or a sample solution. 

The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements. 

•  Repeatability

 Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision . 

• Intermediate precision 

Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment, etc. 

•  Reproducibility 

Reproducibility expresses the precision between laboratories (collaborative studies, usually applied to standardization of methodology). 

• DETECTION LIMIT

 The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. 

•  QUANTITATION LIMIT 

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices, and is used particularly for the determination of impurities and/or degradation products. 

•  LINEARITY 

The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. 

•  RANGE 

The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity. 

• ROBUSTNESS 

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. 

Spectroscopy - Absorption of Different Electromagnetic radiations and Solvent Effects

Introduction

The molecular spectroscopy is the study of the interaction of electromagnetic waves and matter. The scattering of sun’s rays by raindrops to produce a rainbow and appearance of a colorful spectrum when a narrow beam of sunlight is passed through a triangular glass prism are the simple examples where white light is separated into the visible spectrum of primary colors. This visible light is merely a part of the whole spectrum of electromagnetic radiation, extending from the radio waves to cosmic rays. All these apparently different forms of electromagnetic radiations travel at the same velocity but characteristically differ from each other in terms of frequencies and wavelength.

Absorption of Different Electromagnetic radiations

 In absorption spectroscopy, though the mechanism of absorption of energy is different in the ultraviolet, infrared and nuclear magnetic resonance regions, the fundamental process is the absorption of a discrete amount of energy. The energy required for the transition from a state of lower energy (E1) to state of higher energy (E2) is exactly equivalent to the energy of electromagnetic radiation that causes transition.

Fig. Energy transition for the absorption of any electromagnetic radiation

E . .. 1 – E2 = E = hν = h c / λ

Where E is energy of electromagnetic radiation being absorbed, h is the universal Planck’s constant, 6.624 x 10-27 erg sec and ν is the frequency of incident light in cycles per second (cps or hertz, Hz), c is velocity of light 2.998 x 1010 cm s -1 and λ = wavelength (cm)

Therefore, higher is the frequency, higher would be the energy and longer is the wavelength, lower would be the energy. As we move from cosmic radiations to ultraviolet region to infrared region and then radio frequencies, we are gradually moving to regions of lower energies.

A molecule can only absorb a particular frequency, if there exists within the molecule an energy transition of magnitude E = h ν

Although almost all parts of electromagnetic spectrum are used for understanding the matter, in organic chemistry we are mainly concerned with energy absorption from only ultraviolet and visible, infrared, microwave and radiofrequency regions.

Ultraviolet – visible spectroscopy (λ 200 - 800 nm) studies the changes in electronic energy levels within the molecule arising due to transfer of electrons from π- or non-bonding orbitals. It commonly provides the knowledge about π-electron systems, conjugated unsaturations, aromatic compounds and conjugated non-bonding electron systems etc.

 Infrared spectroscopy ( ν 400-4000 cm-1) studies the changes in the vibrational and rotation movements of the molecules. It is commonly used to show the presence or absence of functional groups which have specific vibration frequencies viz. C=O, NH2, OH, CH, C-O etc. 

Nuclear magnetic resonance (radiofrequency ν 60-600 MHz) provides the information about changes in magnetic properties of certain atomic nuclei. 1 H and 13C are the most commonly studied nuclei for their different environments and provide different signals for magnetically non-equivalent nuclei of the same atom present in the same molecule.

In the present chapter, UV-Vis and Infrared spectroscopy have been discussed.

Ultraviolet and Visible Spectroscopy This absorption spectroscopy uses electromagnetic radiations between 190 nm to 800 nm and is divided into the ultraviolet (UV, 190-400 nm) and visible (VIS, 400-800 nm) regions. Since the absorption of ultraviolet or visible radiation by a molecule leads transition among electronic energy levels of the molecule, it is also often called as electronic spectroscopy. The information provided by this spectroscopy when combined with the information provided by NMR and IR spectral data leads to valuable structural proposals.

Principles of Absorption Spectroscopy : Beer’s and Lambert’s Law The greater the number of molecules that absorb light of a given wavelength, the greater the extent of light absorption and higher the peak intensity in absorption spectrum. If there are only a few molecules that absorb radiation, the total absorption of energy is less and consequently lower intensity peak is observed. This makes the basis of Beer-Lambert Law which states that the fraction of incident radiation absorbed is proportional to the number of absorbing molecules in its path.

When the radiation passes through a solution, the amount of light absorbed or transmitted is an exponential function of the molecular concentration of the solute and also a function of length of the path of radiation through the sample.

Therefore, Log Io / I = ε c l

Where Io = Intensity of the incident light (or the light intensity passing through a reference cell)

 I = Intensity of light transmitted through the sample solution

 c = concentration of the solute in mol l-1

l = path length of the sample in cm

ε = molar absorptivity or the molar extinction coefficient of the substance whose light absorption is under investigation. It is a constant and is a characteristic of a given absorbing species (molecule or ion) in a particular solvent at a particular wavelength. ε is numerically equal to the absorbance of a solution of unit molar concentration (c = 1) in a cell of unit length ( l = 1) and its units are liters.moles-1 . cm -1. However, it is customary practice among organic chemists to omit the units.

The ratio I / Io is known as transmittance T and the logarithm of the inverse ratio Io / I is known as the absorbance A.

- Log I / Io = - log T = ε c l

and Log Io / I = A = ε c l

or A = ε c l

For presenting the absorption characteristics of a spectrum, the positions of peaks are reported as λmax (in nm) values and the absorptivity is expressed in parenthesis.

Solvent Effects

 Highly pure, non-polar solvents such as saturated hydrocarbons do not interact with solute molecules either in the ground or excited state and the absorption spectrum of a compound in these solvents is similar to the one in a pure gaseous state. However, polar solvents such as water, alcohols etc. may stabilize or destabilize the molecular orbitals of a molecule either in the ground state or in excited state and the spectrum of a compound in these solvents may significantly vary from the one recorded in a hydrocarbon solvent.

(i)                π -π* Transitions

In case of π Æ π* transitions, the excited states are more polar than the ground state and the dipole-dipole interactions with solvent molecules lower the energy of the excited state more than that of the ground state. Therefore a polar solvent decreases the energy of π Æ π* transition and absorption maximum appears ~10-20 nm red shifted in going from hexane to ethanol solvent.

(ii)             n -π* Transitions

 In case of n Æ π* transitions, the polar solvents form hydrogen bonds with the ground state of polar molecules more readily than with their excited states. Therefore, in polar solvents the energies of electronic transitions are increased. For example, the figure 5 shows that the absorption maximum of acetone in hexane appears at 279 nm which in water is shifted to 264 nm, with a blue shift of 15 nm.

Fig: UV-spectra of acetone in hexane and in water