Career thought's for personal appraisal in Life and Biotechnology

THE FIVE MUSTS OF INTERVIEWING

we know people who seem to get every job they ever interview for, whether or not they’re the most qualified candidate. But most people who interview don’t get the job. This isn’t surprising, given the number of applicants for each opening. But bottom line, there are only five reasons people don’t get the job they’re interviewing for.

Employers are really asking five questions in their interview:

1. Can you do the job?
2. Do you want the job?
3. Will you fit in?
4. Can we manage you?
5. Will you do the job long term?

If you can successfully communicate positive responses to these, then there’s nothing else you need to prove, and you’ll get the job. But the truth is, most people cannot effectively communicate those five simple things.

1. Can You Do the Job?

In most cases, if you’ve earned an interview, you probably can do the job. You’ve demonstrated that you’re qualified, that your education and experience endow you with the requisite “hard skills” to perform and succeed. You generally won’t get an interview if you don’t have the necessary hard skills for the job.

Regardless of your qualifications, however, hard skills alone are never sufficient to get you a job. An interview is rarely about your technical skills and experience, but rather about something else entirely: your “soft skills.” Soft skills have to do with your personality and will be very much on an interviewer’s mind:

• Can you communicate well with others?
• Are you creative?
• Can you adapt to different environments?
• Will you work well with a difficult supervisor?
• Will you do more than is expected of you?
• Can you work in a team?
• Are you willing to learn?

Many people in the work world have mistaken ideas about meritocracy and the importance of hard skills in the end. Here are some meritocracy misconceptions you may be ingrained with:

• Your past achievements guarantee your future success.
• Going to a good school means success in life.
• Having good grades makes you smarter and more likely to succeed.

Yes, all of this matters to some degree, but make no mistake: quite often candidates with less impressive qualifications get more offers because they do a better job selling their soft skills in the interview. Regardless of what you’ve done in the past, employers are really only concerned about what you can do in the future.

Several years ago I hired someone from a unionized position in a school system to start a division within our company. This position required creativity, multitasking, and working long hours. Although this candidate met the job requirements, hiring her turned out to be a gigantic mistake for the simple reason that she was unable to adapt to a new environment.

Shortly after joining us, she started asking about various procedures she was accustomed to that she felt should be in place. She insisted that everything be documented. She was upset by the company’s lack of protocols and process and the absence of formal break times. She was used to structure and reporting hierarchies that were foreign to our still-formative company.

Her expectations weren’t unreasonable, and had our work environment been similar to her previous one, she would have had the perfect soft skills for the job. But our company’s unstructured environment was too different, and her soft skills did not mesh with it.

When I got my last dog I named him Badger, because I admire the characteristics of badgers:

• Badgers can work alone, but also in groups (badgers have even been known to hunt with coyotes).
• Badgers hunt all night and day.
• Badgers are completely fearless (they’ll eat larvae from a bee’s nest).
• Badgers are fierce, will protect themselves, and are generally unafraid of other animals.
• Badgers are thick-skinned (literally)—so much so that it’s difficult to pierce with even a bow and arrow. The looseness of badgers’ skin also allows them to twist and turn on their attackers when held.
• Badgers will attack and eat venomous snakes.
• Badgers will hunt in the open and will even fight people.
• Badgers are intelligent and can use tools.

As with badgers, you’ll be served well by being versatile, easily adapting to a new environment, and having a strong work ethic. Because when employers consider you for a job, they’re mainly looking for a good fit with the company.

Employers are also deeply interested in your level of productivity. Some people show up to a job, work hard, and accomplish all their tasks for the day, while others work more slowly and less productively. Obviously, if you had a business to run, you’d be most interested in hiring the people who show up to work and get a lot done.

When prospective employers perceive you as extremely productive, you immediately stick out as someone hirable. People who work hard and stay focused rarely lose their jobs. So how do you convey a high productivity level?

• Be prepared to talk about your experience. When asked about your experience, don’t just list your work history. Tell brief stories that address the qualities the employer seems to be seeking for the job: attention to detail, getting along well with others, making the employer money, working hard.
• Keep answers about your experience to under two minutes. Stories longer than a minute or two are likely to bore your interviewer, an obvious negative for you. Most interviewers prefer succinct answers that get to the point quickly while still conveying your personality.
• When talking about your experience, detail the results you’ve gotten in the past. Employers are more concerned with results than your past duties. Include specifics about what you achieved in your previous employment and emphasize the results you could produce for your new employer as well.
• Appear genuinely interested and enthusiastic about your profession and what you do.  Employers have a choice of many candidates, so you need to appear focused and proficient in the subject matter relevant to the job in question. People with a profound interest in and passion for what they do are always preferable to those who simply do a job. After all, whom would you want to hire, someone with a passing interest or someone who takes the work seriously? Talk about seminars you’ve attended on your own time, books you’ve read, and other extracurricular activities related to the job. Make sure the employer gets the impression that you’re highly committed.
• Remember that you’re marketing a product — yourself. When you interview and describe your experience and qualifications, you’re presenting a product and need to make your proficiencies stand out; make it clear you can do the job and do it well. Use strong, confident language and make eye contact. Sell yourself. Employers don’t care that you have bills to pay or want a prestigious position; they’re looking only for the best possible product to fill their needs.

2. Do You Want the Job?

A surprising number of candidates show up for an interview when they don’t really want the job. Many people seem to either think they’re above the job, see the job they’re interviewing for as a steppingstone to something better, or are interested just in the money. That attitude comes through loud and clear.

Make absolutely sure the interviewer realizes you’re there to do the job you’re interviewing for, and that this is the job you want. If the interviewer senses for a moment that you’d prefer doing something else, chances are you’ve blown it.

I cannot tell you how many people I’ve interviewed in the past who told me they planned on doing something else in a few years. Why would I hire them? I’d rather hire someone who wanted the job than someone just passing through. I’ve had jobs for gardeners, for example, and been astonished when guys with Ph.D.s showed up with stories about a slow employment market, expecting me to hire them. I’d have to be out of my mind to hire a Ph.D. as a gardener who was interviewing because of a bad job market:

• He wouldn’t be committed to the job, and he’d want to do something else.
• He’d resent the job and what he’d be paid to do it.
• He’d feel the job was beneath him.

Employers hire people who really want the job. An employer wants to feel good about giving someone a job. When I hire a gardener, the best candidate has done gardening for his or her entire career. This person understands plants and how to do the work and enjoys doing it. I don’t want to hire someone who doesn’t really want the job. People who like their jobs almost always are better at it than those who don’t.

When people are trying to judge whether or not you want the job, they’re almost always asking whether your heart is in what you are interviewing for. Many people interview for jobs their heart just isn’t in. Employers can tell this about you very quickly. So how do you convey that you really want the job?

• Look, act, and dress professionally. You want to come across as polished, even flawless. People who want jobs are on their best behavior for interviews. This is a basic sign of respect that shows the employer you want the job.
• Bring your resume, samples of your work, and a written list of references with you. People who want the job come equipped with this information. You need to be prepared to talk about your experience in as much depth as the employer wants. You also want to show your interviewer that you have plenty of materials to back up your qualifications. Bringing samples of your work and a list of references shows confidence and a forward look.
• Learn as much as you can about the employer and your interviewers.Learn everything you can about the company you want to join. Plan to spend at least a few hours reading up on the company before you even set foot in that interview. Read their mission statement, company history, and anything currently in the news. Show them you’re truly interested in working there. If you have the names of your interviewers ahead of time, read up about them too.

3. Will You Fit In?

Fit is of massive importance to employers. It’s one of the main things interviewers screen out, and they do it fast.

If you show up to interview with a fitness company and you’re 55 years old and 100 pounds overweight, do you think you’ll fit in? You won’t stand a chance of getting the job. You know it and the company knows it. You just won’t fit in.

When I was practicing law, I once represented a small trucking company that had been sued by a woman who’d applied to be a driver. That trucking company in a rural town was made up of all men and was a macho type of place. She was (predictably) not hired and sued the trucking company. The basis of her lawsuit was that she hadn’t been hired because she was a woman.

When the case got to trial, you could see the snickers of the jurors, judge, and just about everyone else involved but the woman and her attorneys. Clearly, the woman would not have fit in with the group of rough truckers. Despite evidence that she might have been as qualified as the male truckers who’d been interviewed, it took the jury less than 20 minutes to return a verdict against her. The woman had not been hired because she was not a good fit.

Fit is incredibly important to employers. You need to appear to be a person who’ll fit in.

• How you dress for your interview is a factor in whether you fit in.
• What employers pick up about your political leanings is a factor in whether they think you fit in.
• Your sexual orientation (and how evident you make it) is a factor in whether you fit in.
• Your religion is a factor in whether you fit in.
• Your sex is a factor in whether you fit in.
• Your age is a factor in whether you fit in.
• Your race is a factor in whether you fit in.
• Your interests outside of work are a factor in whether you fit in.
• Your general personality is a factor in whether you fit in.

If you don’t think this stuff matters and that employers are open-minded, you’ve got another thing coming. It doesn’t matter if the company calls itself an equal-opportunity employer. Employers care a ton about whether or not you fit in, and they take it incredibly seriously.

Several years ago 60 Minutes featured a young technology company made up mainly of young college graduates. These kids were all sleeping at their desks, bringing their pets to work, eating their meals there. I remember watching this show with a couple in their early 50s.

“Dear Lord!” one of them said. “Who on earth would do that?”

Now it just so happens that these two people were unemployed. Do you think someone in his 40s or 50s could show up at this company and get a job? Absolutely not. They wouldn’t fit in. Intelligence, background, and experience would simply not matter when it came right down to it. A more important question would be was that person willing to sleep at his desk? Work seven days a week? Share interests with his co-workers?

All of this matters.

Remember, the interviewer needs to like you. In order for this to happen, you must appear to fit in with the culture of the organization. Each organization has its own culture. You need to assess this culture quickly and do your best to appear as if you’d fit in. At the same time, be honest with yourself. Would you be happy in this environment? A good fit works both ways.

Before any interview, ensure that you understand the values of the company. These values are often on the website or in corporate brochures. In the interview, it’s always a good idea to refer to some of these values to demonstrate a good fit. How?

• Don’t talk about conflicts you’ve had in past positions. Any sort of conflict is a red flag and signals a questionable fit. Leave it out, whatever it was.
• Avoid divulging personal details like your religion or political affiliation. While this might help you in some instances, you simply have no way of knowing for sure how this will go. It’s better not to bring it up at all.
• If asked illegal questions (about your race, age, ethnicity, and so forth) be careful not to offend the employer; answer as gracefully as possible. A woman   who’s asked her age, for example, can demur with humor with something along the lines of “A lady never tells her age.” This sidesteps the question with a minimum of fuss. Be careful with these sorts of questions should they come up. Although you may decide you don’t want to work for a company that doesn’t respect lawful hiring practices, you also don’t want to make a bad name for yourself in the industry by reacting poorly.

4. Can We Manage You?

In just about any interview, you’ll be asked why you left your previous employer, and the one before that, and so on. Employers want to determine whether you’re manageable. What does it mean to be manageable? For most employers it comes down to being good at taking orders.

Often when people are asked this question, they start to complain about their past employers. They talk about how much they disliked their supervisor, the bad morale in the company, and so forth. If your reply is the least bit negative toward supervisors and others, you’ll come across as someone who cannot be managed.

I’ve heard people in interviews say they quit because they didn’t think it was fair that their boss asked them to run an errand during work, or because they were given work they thought was beneath them. Say anything like this and you’ll appear unmanageable.

Employers expect you to show up on time, be ready and willing to work, and be happy following orders as well as work independently. If you don’t want to be accountable to someone else or follow orders, then you shouldn’t be working for someone else. Ultimately, though, everyone works for someone else.

Being perceived as someone manageable means that you’re seen as someone who puts the organization and job first. If you’re asked to do something, the employer needs to be confident you’ll do it and make it a priority. An employer wants to believe that your career is your priority, and following orders is part of that.

Concerned about things like work-life balance? That’s fine; just keep it inside. The employer needs to believe that what matters to you is not work-life balance but your job. When it comes to working for just about any employer, it is not, regrettably, remotely about you—it’s about the employer.

Many people get distracted when they get older and end up getting interrupted more often. It becomes harder for them to focus as much on work, and their lives away from the job take over. Time away from work, devoted to family, sports, children, activities, church, and vacations, moves up in priority. Many older job candidates discuss personal commitment issues during the first interview. This is a mistake..

People who get distracted by tasks away from the office are effectively having their careers and time managed by someone or something else. This is not what an employer wants to see or hear. An employer needs to believe that your number-one priority is your career and job. People whose careers are their priority are infinitely more manageable than those who have other priorities.

Being manageable also means being flexible. For example, when employers ask whether you’d be willing to move, they’re interested in what you’re willing to give for the job. You want to land that position? Demonstrate that you’re willing to do whatever it takes to get the job done.

• Show yourself to be someone who’ll do what’s asked. People who question orders are considered unmanageable. If you’re applying for a position that requires that you take orders from someone—which is most often the case—you must appear willing to follow those orders.
• Always speak positively about your former employers. Negative comments give the impression that you’ll question authority and have “anger issues,” even if your negative feelings were justified. You need to be seen as affable and able to work with different personalities.
• Talk about positive performance reviews that you’ve received if the opportunity arises. People generally get positive performance reviews only if they’re doing a good job and following orders.

5. Will You Do the Job Long Term?

Employers are extremely interested in whether or not you’ll continue with the company, and in that current position, over the long term. Anyone can show up for an interview—or the first day of work. Not everyone will continue to show up month after month and year after year. That’s the kind of person employers want to hire.

Many people are dabblers; they never commit to one thing for the long haul. How many projects have you, for example, started, such as a hobby, exercise routine, or diet, only to stop a short time later? But some people keep going and push through, even when the going gets tough. They don’t allow themselves to get distracted. They’re the ones employers want to hire.

It’s expensive for employers to hire people. They spend valuable time seeking out candidates. Then there’s the expense of training someone and arranging setups with IT and other departments. And if the person leaves, there’s the drag on morale to contend with. For these reasons, if employers don’t think you’ll do the job long term, why hire you? You’re just going to waste their time and money.

If you have stellar qualifications and are attempting to get a position with a not-so-prestigious company, you may actually find it difficult to get hired. Why? Far from being impressed with you and your qualifications, the company may think you’ll leave when something better comes up. They may also think you want higher compensation than they’re willing to give and will move on for more money should the opportunity arise. Employers don’t want to risk that, so you might find in some instances that you can’t even get an interview.

Similarly, if your resume is littered with various job changes, employers will reason that you’ll change jobs again at the drop of a hat. Lots of people move between jobs like this, and generally each successive job becomes more and more difficult for them to find.

Another issue for employers comes up if you’re attempting to enter a new field. If you’re interviewing for a position that doesn’t appear to be the sort you’ve had before, the employer may worry that you won’t stick around. Because you’re trying something new, you might turn out not to like it. Employers are taking a risk that you’ll both excel in the position and enjoy it enough to see it through and stay with the company.

If you do have several job changes on your resume or if you’re making a career change, you need to communicate to employers that you’re capable of sticking a job out. An employer wants to know that this position represents something significant to you, something you’ll commit to for the rest of your career.

Remember these concepts when you interview:

• Employers want people committed to the geographic area they’re in. They want to know you’re connected and set to stay. Particularly if you’re planning to move to a new area for the job, you need to play up any connections you have there to demonstrate that you’ll stick around.
• Employers want people committed to the profession they’re in. Be sure to come across as someone who wants to do the job for many years, if not your entire career. Even if you’ve changed companies, you need to explain your experience in such a way that it all appears connected and part of your career path.
• Employers value stability in both your career and your life. A home, a mortgage, and a family are all signs that you’re stable and not going anywhere. When an employer learns that you’re “settled down,” this signals that you’re committed and are likely to continue with the company over the long term. If there’s a chance your interviewer thinks you’re a flight risk, don’t be afraid to indicate you have a stable home life.

You can read entire books about interviewing, but when it comes right down to it, what employers care about are the five questions presented here: (1) Can you do the job? (2) Do you want the job? (3) Will you fit in? (4) Can we manage you? (5) Will you do the job long term?

Prepare the answers to these questions when you interview, and your chances of landing the job will increase exponentially.

Instrumentations in Biotechnology!!!

Biophysical and Biochemical Techniques:- 
Photometry; Nuclear magnetic resonance spectroscopy; Electron spin resonance spectroscopy; Atomic absorption spectroscopy; Chromatography; Electrophoresis; Centrifugation; Mass spectroscopy; Spectrophotometry

Biochemistry is not pure, but an applied discipline and is both analytical and quantitative to explain cause and effect relationship in molecular terms. Analysis means getting to bottom of things. It is used only when combined with synthesis i.e. combining together all pieces obtained by analysis followed by extrapolation of observations to understand the whole system.

Qualitative analytical biochemistry is concerned mainly at molecular level and at times at electronic level as well. Quantitative analysis is concerned with measurement of concentration of constituents identified by qualitative analysis.

Separation and identification of particular compound from complex conglomeration in biological tissues is accomplished by various techniques such as chromatography, electrophoresis, centrifugation, mass spectroscopy, ultraviolet, visible and infrared spectrophotometry, nuclear magnetic resonance and electron spin resonance spectroscopy and so on.

Photometry is one of the powerful tool of analysis and is done at various wavelengths in the ultraviolet, visible and infra red regions of the electromagnetic spectrum. The technique is used both for identification of compounds and their quantitation. Absorption of electromagnetic radiations by compounds is highly specific phenomena. Different wavelengths have different quanta of energy. A compound will absorb only that wavelength of radiant energy which has just the same amount of energy that is needed by the compound for excitation to higher energy level. Energy absorbed in the ultra violet and visible region of the spectrum causes excitations in the electronic state of the compound to a higher level. Since this region contains high amount of energy, the absorbed energy is also capable of causing excitations in the molecular vibration and rotation states, therefore in this region we obtain absorption bands rather than line spectra. The energy requirement for molecular excitations to higher electronic and rotational vibrational levels is fixed and depends upon the structure of the molecule and only that particular wavelength having the same amount of energy as needed by the molecule would be absorbed. This wavelength of absorption is characteristic property of the molecules and is used for their identification and since amount of radiation absorbed at that wavelength is proportional to the concentration of light absorbing molecule, the technique is widely used for quantitation of the molecule.
In the infra red region, where quanta of energy in the radiations is lesser, the absorbed radiations cause excitations in the vibrational and rotational levels of the molecule. Pure rotational spectra arise in the far infra red region and are of little use to biochemists. Absorption spectra in this region are mainly used to determine specific groups in compounds.

More information about structure of many molecules can be obtained by nuclear magnetic resonance spectroscopy. In this technique the nuclear spins of molecules are excited by placing the sample between the pole pieces of strong magnet and applying variable rotating radio frequency to cause resonance. Depending upon the nuclear spin different nuclei resonate at different frequency. This property is used to distinguish and estimate different compounds having different nuclear conformation. Excellent analytical studies of compounds having unpaired electrons and free radicals is achieved by electron spin resonance spectroscopy where radiations in the microwave region are used to cause electron resonance for the analysis.

Many compounds, especially those having multiple conjugated double bonds with high degree of resonance stability, after absorbing electromagnetic radiations at a particular wavelength, exhibit fluorescence and the latter is used again both for quantitative and qualitative analyses. Atomic absorption spectroscopy is yet another powerful tool to study certain elements such as Na, K and Ca, which otherwise cannot be detected and estimated by classical methods. Mass spectroscopy sorts out ions on the basis of differences in their mass/charge ratio and is widely used to analyze various compounds including oligopeptides.

Centrifugation has been used to separate subcellular components and macromolecules by sedimenting them under enhanced and varying gravitational force. The technique is especially valuable for determination of molecular weight of macromolecules as the sedimentation rate is dependent upon molecular dimension and size.
Isotopes, in particular the radioactive ones have been employed in studying various metabolic pathways. Isotopes differ in physical property but have the same chemical behavior. Radioactive isotopes are easily detectable and are used as tags to decipher metabolic pathways, to study the kinetics of enzyme reactions and in diagnosis and therapy of many types of cancers.

Other routinely used analytical tools are different types of chromatography and electrophoretic techniques. Details of these techniques are given in different sections to follow

Bio-analysis of information processing molecules, DNA and RNA has lead to clearer appreciation of the connection between base sequences in the DNA, and how specific proteins are formed thus allowing to understand how cells differentiate during development. Artificial synthesis of DNA, using polymerase chain reaction and methods for multiplying and transferring DNA to unnatural recipients in genetic engineering have solved many hitherto unknown mechanisms and problems. All these advances that we see in the field of biological sciences owe to various physical and chemical, qualitative and quantitative techniques that have been employed by researchers.

Chromatography

Chromatography is a separation technique, which involves the distribution of components of a mixture between the stationary phase and mobile phase. Separation of components in a mixture is based on the fact that the rate of migration of individual component through adsorbent is directly related to the partition of that component between the two phases.

Column chromatography:

Organic compounds with the help of Column Chromatographic technique are separated .Column chromatography is one of the most useful methods for the separation and purification of both solids and liquids. This is a solid - liquid technique in which the stationary phase is a solid & mobile phase is a liquid. The principle of column chromatography is based on differential adsorption of substance by the adsorbent.

Intermolecular forces, which vary in strength according to their type, make organic molecules to bind to the stationary phase. The stronger the intermolecular force, the stronger the binding to the stationary phase, therefore the longer the compound takes to go through the column.

Two substances under test are mixed and the solution is poured through the column containing the adsorbent. Different components will be immediately adsorbed and fixed in a layer, some hold fast, while others are slow. The process of desorption occurs when the column is irrigated with a solvent. This process is called elution. As a consequence of desorption the components, which are held loosely, travel faster down the column than those held strongly by adsorption force. Thus as a result of continuous irrigation with solvent, different components can be separated from one another in the form of distinct bands across the length of the adsorbent column.

Intra-molecular hydrogen bonding is present in ortho- nitro phenol. This is due to the polar nature of the O-H bonds which can result in the formation of hydrogen bonds within the same molecule. But in para-nitro phenol, inter molecular hydrogen bonding (between H and O atoms of two different para-nitro phenol molecules) is possible. As result of inter molecular hydrogen bonding para-nitro phenol undergo association that increases the molecular weight, whereby decreasing volatility.

Para nitro phenol with inter molecular hydrogen bonding:

Ortho nitro phenol with inter molecular hydrogen bonding:

Procedure:

1.      Preparation of the column:

Ø  Place the column in a ring stand in a vertical position.

Ø  A plug of glass wool is pushed down to the bottom of the column.

Ø  Prepare slurry of silica gel with a suitable solvent & pour gently into the column.

Ø  Open the stop cock & allow some solvent to drain out. The layer of solvent should always cover the adsorbent; otherwise cracks will develop in the column.

2.      Adding the sample to the column:

Ø  Dissolve the sample mixture in a minimum amount of solvent (petroleum ether).

Ø  Remove the solvent by placing the mixture in a rotary evaporator at a low temperature.

Ø  Place the dry powder on a piece of weighing paper and transfer it to the top of the column through the funnel.

3.      Developing the chromatogram:

Ø  Attach a dropping funnel filled with petroleum ether on to the column.

Ø  Add petroleum ether continuously from the funnel to the top of the column.

Ø  Open the stopcock carefully.

Ø  The components of the mixture run down the column forming two separate yellow bands.

4.      Recovering the constituents:

Ø  Continue running the petroleum ether till both the bands are eluted out separately.

Ø  Collect the constituents in two different R.B flasks. (Ortho nitrophenol is obtained first, followed by para nitro phenol.).

Ø  Evaporate the solvent by placing the mixture in a rotary evaporator.

 Books:

1.      Frederick George Mann, Bernard Charles Saunders, Practical organic chemistry. 1960

2.      Solomons, Fryhle. Organic Chemistry, 8th Edition Wiley-India, 2006.

3.      The systematic identification of organic compounds by Ralph L Shriner 8th edition

Spectrophotometry

Spectrophotometer measure the absorbance of the sample at different wavelengths, find out the unknown concentration of the sample through Beer - Lambert's law.

 A spectrophotometer is a photometer that can measure the intensity of light as a function of its wavelength. Single beam and double beam are the two major classes of spectrophotometer. The single beam spectrophotometer measures the relative light intensity of the beam before and after a test sample is inserted and the double beam spectrometer compares the light intensity between two light paths, i.e. one path containing the relative light intensity of the beam before and after a test sample is inserted and the instruments are easier and more stable whereas the single beam instruments can have a larger dynamic range and are optically simpler and more compact. Spectral bandwidth and linear range of absorption measurement are the important features of spectrophotometers.

In transmission measurements the spectrophotometer quantitatively compares the amount of light passes through the reference and test sample and for reflectance it compares the amount of light reflects from the reference and test sample solutions.

Many spectrophotometers must be calibrated before they start to analyse the sample and the procedure for calibrating spectrophotometer is known as "zeroing." Calibration is done by using the reference substance, i.e. the spectrophotometer is set as a baseline value, so the absorbencies of all other substances are recorded relative to the initial "zeroed" substance. The spectrophotometer then displays % transmissivity (the amount of light transmitted through the substance relative to the initial substance).

The major sequence of events in spectrophotometry is as follows:

1.      The light source shines through a monochromator.

2.       An output wavelength is selected and beamed at the sample.

3.       A fraction of the monochromatic light is transmitted through the sample and to the photo-detector.

 

Single beam spectrophotometer:

Spectrophotometry deals with visible light, near UV and near IR. Especially for IR spectrophotometers, there are spectrophotometers that use a Fourier transform technique to acquire the spectral information quicker in a technique Called Fourier Transform Infrared

Different Types of Spectrophotometers: 

A. Single Beam:  In this all the light passes through the sample and to measure the reference intensity of the incident light the sample must be removed so that all the light can pass through. This type of spectrometer is usually less expensive and less complicated.

B. Double Beam: In this type the light source is split into two separate beams before it reaches the sample. From these two beams one is used for reference and the other passes through the sample. This is advantageous because the reference reading and sample reading can take place at the same time.

C. Visible Light (400-700 nm): Visible region spectrophotometers vary in accuracy. These spectrophotometers can use incandescent, halogen, LED, or a combination of these sources.

D. Ultraviolet Light: UV spectroscopy is most commonly used for liquids, but can also be used for gases and even solids. Samples are placed in a cuvette, which are made of plastic, glass, or quartz. Plastic and glass absorb UV, so they can only be used for visible light spectroscopy.

E. Infrared Light: Infrared spectroscopy is used to study molecules and the vibrations associated with their structures. Due to the varying energies associated with each wavelength different chemical structures vibrate in different ways in response to different wavelengths. For example, mid-range infrared tends to cause rotational vibrations, while the near infrared (higher energy) tends to cause whole molecule harmonic vibrations like stretching, and rocking.

Beer - Lamberts law:

Diagram of Beer-Lambert absorption of a beam of light as it travels through a cuvette of width l.  

Beer Lambert’s law is the linear relationship between the absorbance and concentration of the absorbing sample, i.e. there is a logarithmic dependence between the transmission (or transmissivity), T, of light through a substance and the product of the absorption coefficient of the substance, α, and the distance the light travels through the material (i.e. the path length), l. The absorption coefficient can be written as a product of molar absorptivity of the absorber, ε, and the concentration c of absorbing species in the material, or an absorption cross section, σ, and the (number) density, N, of absorbers.

For liquids, these relations are usually written as:

Whereas for gases, these relations are written as:

 

where,

I₀=intensity of the incident light

I=intensity of the transmitted light

σ=cross section of light absorption by a single particle

N=density of absorbing particles 

The transmission (or transmissivity) for liquids in terms of absorbance, is defined as:

The relationship between absorbance (A) and percent transmittance (%T) is also quantitative and this can be written as:                                                   

 Percent transmittance is  

Whereas, for gases, it is usually defined as:

This shows that the absorbance becomes linear with the concentration (or number density of absorbers) according to:

and

Thus, if the path length and the molar absorptivity are known and the absorbance is measured, the concentration of the substance can be deduced.

According to the Beer-Lambert Law, absorbance is proportional to concentration, so that at dilute solutions a plot of concentration vs. absorbance would be straight line, but the Law breaks down for solutions of higher concentration, and so you might get a curve under those circumstances.

 Applications of a Spectrophotometer

1. It directly used to measure light intensity at different wavelengths

2. It is used to determine the unknown concentration of solution

3. Spectrometers can be used to determine the equilibrium constant of a reaction involving ions

 Books:

1.    Modern Instrumental Methods and Techniques - John Wiley & Sons, West Sussex, Skoog D A

2.    Instrumental methods of analysis - Willard, Merritt L L, Dean J, Sette P A

3.    Vogel's textbook of quantitative chemical analysis - Arthur Israel Vogel, John Mendham