Chemical Wonders: Experimental Techniques (Part 3) - Chemistry in the Infrared

Infrared & Raman Spectroscopies

Infrared spectroscopy is an invaluable tool for chemists, and is applied in fields from astronomy to forensics. It takes advantage of the transitions between vibrational energy states of molecules, and in order to be classed a 'IR active,' there need to be a change in the charges of the dipole moment. The other mode of vibration, called Raman spectroscopy, deals specifically with the change in polarizability. Both IR and Raman spectroscopy are forms of vibrational spectroscopy. The IR region ranges from 20 cm^-1 to 14 000 cm^-1 (called near IR).

The wavenumbers of molecular vibrations

When a molecule is exposed to infrared radiation, its covalent bonds vibrate and stretch (think of it like stretching a spring), when this occurs the molecule undergoes harmonic oscillations. The energy levels of these vibrations are given by:

E_v = (v + ½)hv - (v + ½)² hvx_e      (E_v is in J, joules)

where v = vibrational quantum number; h = Planck constant; v = frequency of vibration; x_e  = anharmonicity constant. At an energy level, where v = 0, is the zero point energy of the molecule. When dealing with a transition from the vibrational ground state, to the first excited state, the motion of this molecule is approximately one of a simple harmonic oscillator:

E_v = (v + ½)hv 

When considering a hypothetical diatomic molecule, say, XY you would find that the vibrational frequency is dependant on two factors: the mass of atoms X and Y; and the force constant (k) of the bond. The constant, k, is determined by the strength of the covalent bond or in other words the stiffness of the "spring." 

• Diatomic molecules with where X and Y similar masses, they approximately contribute equally to the molecular vibration.
• In molecules where X and Y have significantly different masses, the lighter atom moves more than the heavier one.

The reduced mass,𝜇, is the quantity that describes the mass of the oscillator so that it can more accurately reflects the relative masses of X and Y. This relationship is given by:

1/𝜇 = 1/m_x + 1/m_y           OR           𝜇 = m_xm_y / m_x + m_y 

The fundamental absorption of the molecule is the transition from the ground state to the first excited state. This relationship can be given by:

v = 1/2𝜋 √k/𝜇

where: v = the fundamental vibrational frequency (Hz); k = force constant (N m-1);  𝜇 = reduced mass; 𝜇 = reduced mass. Using these definitions, we can define the relationship of the absorptions in IR spectra in relation to the wavenumber, it can be defined as below:

 = 1/2𝜋c√k/𝜇 

where  = wavenumber (cm-1); c = speed of light  = 3.00x10¹⁰ cm s^-1.

Characteristics of IR spectra

The IR spectra produced from using a device such as the Fourier transform infrared spectrometer (FT-IR, shown above), can be separated into two main regions: the fingerprint region, which occurs in bands in the regions below 1500 cm^-1, which arise from single bond stretching nodes, vibrations within the molecule and deformations in the molecular structure. Any absorption wavelength found above this region is typically what is used to identify key functional groups on the compound in question. The fingerprint region on the other hand is used to identify the characteristic signature of the compound, as the bands in these regions are specific to the overall structure of the molecule.

You can find a table of these functional groups here.

I mentioned that the IR spectra ranges from 20 cm^-1 to 14 000 cm^-1 (called near IR), however, in the lab, IR spectrometers typically use the range from 400 to 4000 cm^-1, dubbed the 'mid-IR' section of the spectra. Data produced from the machines, like the FT-IR produce spectra in which the transmission (at arbitrary values 0 to 100%) against the wavelength of the IR bands. Samples in a typical FT-IR machine can be recorded using samples in gaseous, liquid or even solid samples. Samples in different states need to be prepared in different ways and result in slightly different IR spectra. 

Solids are typically prepared in a mull, mixing the solid with an organic oil, or it is pressed into a disc by grounding it with a an alkali metal halide (such as KBr). These forms of preparation affect the IR spectrum: the disc preparation reduces the range observed by the spectra, as it is transparent from 4000 to 450 cm^-1, while NaCl is from 4000 to 650 cm^-1. More modern machines utilise diamonds, the accessory known as a diamond attenuated total reflector (ATR), which allows us to avoid using mulls or discs.

Raman Spectroscopy

IR and Raman spectroscopy are two techniques which can be used together, and in 1930 Chandrasekhara V. Raman (pictured left) won the noble prize in physics. He discovered that radiation is scattered when a molecule is exposed to a frequency, v₀, even though there is no change in frequency. This is called Rayleigh scattering, which is also responsible for the blue colour of the sky. A small amount of scattered radiation has frequencies of v₀ ± v, where v is frequency of the vibrating section of the molecule. This is known as Raman scattering. It is actually quite an unsensitive because a only a small range actually undergoes Raman scattering. Improvements have been made by using Fourier transformation (FT) techniques. 

Raman spectroscopy is particularly useful because it utilises wavelengths below the normal IR spectroscopy range, this in turn, allows chemists to observe the vibrational nodes found in metal-ligand bonds. Coloured compounds rely on laser excitation that coincide with the absorption wavelengths in the electronic spectrum, known as resonance Raman spectroscopy. Utilising resonance enhancement allows for more clearly defined lines.

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Don't forget to check out my Patreon,  if you like the content I'm putting out:

 https://www.patreon.com/MrElement 

Links provided bring you to some of the info I used from the web. the first/second-year university textbooks:

• "Inorganic Chemistry," 4ED, by Housecroft & Sharpe, Pearson Ed. Ltd., 'Chapter 4 - Experimental techniques,' pgs 90-98. You can buy it here.
• "Fundamentals of Analytical Chemistry," 9ED, Skoog & West, Pearson Ed. Ltd., 'Chapter 28 - Atomic Spectroscopy,' pgs 774-775, 790-799. You can buy it here.

Chemical Wonders: Experimental Techniques (Part 2) - Identifying Elements and Ions in a Compound

When identifying compounds in a lab, you utilise different techniques to get an accurate data output for the compound in question. This part will focus two important parts of experimental chemistry (you may recognise some things from high school chemistry): elemental analysis using techniques such as combustion, atomic spectroscopy, and mass spectrometry, which utilises the ionization of the molecule.

Mass Spectrometry (MS): Separating ions

There are a variety of mass spectrometry techniques which all share one thing in common: separating ions from the compound, whether they are atomic or molecular, and separating them according to their mass-to-charge ratio (m/z). I'll only cover two classic techniques, such as Electron Ionization (EI-MS), and electrospray (ESI) techniques. Other techniques include: fast atom bombardment (FAB) and as well as matrix assisted laser desportion ionization time-of-flight (MALDI-TOF). These techniques are familiar to all chemists and biochemists as extensively useful tools in analytical chemistry,

Electron Ionization (EI) Mass Spectrometry

Electron Ionization or electron impact mass spectrometry would probably be the most familiar to you if you did high school chemistry. This technique is known as a 'hard technique' as it involves bombarding the analyte with high-energy electrons (  ≈70 eV, electron volts), causing the molecule to fragment thus producing ions. It is widely used for analysing organic compounds, but becomes increasingly more limited as the molecular mass of a compound increases (Mr < 1500). This technique can't be used on ionic compounds, and the analyte must be stable when vaporized (if it isn't a gas at 298.15 K). As such this technique is limited to vaporized substances. In general, the molecular breakage can be represented generally as:

M(g) + e^-_{(high energy)} -------------> [M]⁺ (g) + 2e- _{(low energy)}

Using a high-energy electron stream is essential because it is used to break the high covalent bond energies present in the analyte. Two ions are produced, the parent ion and the ion. The [M]⁺ (g) cation is a radical and is written as [M]^●+ which are highly reactive. Fragmentation of molecules is always considered a 'hard technique.' After bombardment with electrons, they pass through a magnetic field, where the positive ions are deflected into a detector. Deflection is entirely dependent on the size of the m/z ratio: a larger m/z value leads to a greater radius of deflection. For ions with a value of z = 1, the m/z value is the same as the molecular mass; if z=2, the m/z ratio is half that of the molecular mass of the ion, and so on.

However, due to being restricted to molecules with relatively low molecular and low energy of vaporization, most ions have z=1. The mass spectrum is plotted so that m/z lies on the x-axis, and the y-axis is the relative intensity of the fragments, arbitrarily set on a scale of 0 to 100%. The final output depends on isotopes of elements that may be present in the molecule too, leading to an observation termed peak envelopes. The device can be summarised below:

Electrospray Ionization (ESI) Mass Spectrometry

This technique is widely used in molecules that have relatively high molecular weight (Mr ≤ 200 000). In contrast with EI mass spectrometry, this technique can be used with ionic substances, where singly and multiply charged ions can be observed in the resultant mass spectrum. This gives give it an advantage over the EI technique. It is also termed a 'soft' technique, as it involves the injection of a sample dissolved in a volatile (such as MeCN or MeOH which are easily vaporized) solvent, which is then sprayed (at 1 atm) into an applied electrical potential. The potential between the original point of injection to the counter electrodes is ~3000 V in positive ion mode. As the ions move toward the counter electrodes, the solvent evaporates and  the gas-phase ions produced eventually hit the mass analyser. Peaks produced that are one mass unit apart reveal an ion is singly charged; on the other hand if they are half a mass unit apart, it is doubly charged, and so forth.

Like FAB and MALDI-TOF, neutral molecules are converted into positive ions with the help of H⁺ and Na⁺. As a result, an aggregate may be produced, generally they either result in a [2M+Na]⁺ and  [M + MeCN + H]⁺.

Elemental and Compositional Analysis

Combustion

For a quantitative analysis of carbon, hydrogen and nitrogen containing compounds, it is possible to fully combust the compound and using the reaction stoichiometry, reach a conclusion for the composition of an analyte. It is done when a known mass of a substance (e.g. 2 to 5 mg) is sealed in an aluminium or tin capsule. This is placed in a fully automated analyser, where it is injected into a pyrolysis/combustion tube and heated to 900 ℃ in a pure oxygen environment. For compounds containing C, H, and N, they are oxidised into CO₂, H₂O and nitro-oxide gases, respectively.

These are then moved using a carrier gas (He) into a copper chamber, where nitro-oxides are reduced into N2 gas and excess O₂ is removed. From here, the CO₂ and H₂O gas mixture is separated and then moved into and analysis chamber, where separated using a type of gas chromatography, which doesn't have a mobile phase (refer to the previous part of the series). The separated gases are then detected using a thermal conductivity detector, the detection process takes about five minutes. The accuracy the recorded amounts of C, H and N present in the compound is about <0.3%.

More modern machines can determine the amount of O and S present, where they are converted into SO₂ and CO to CO₂, respectively.    

Atomic Absorption Spectroscopy (AAS)

This technique is used for determining the quantitative amount of metal but utilising the absorption and emission spectrum of elements. For example, the emission spectrum of hydrogen consists of very sharp lines, each of which corresponds to electronic transitions between high/low energy levels. On the other hand, the absorption spectrum of hydrogen occurs when it becomes irradiated, each element has its own absorption and emission spectrum. AAS is a common type of spectroscopy which uses a hollow cathode lamp calibrated to a given wavelength (specific to a transmission from one energy state to another), which irradiates the analyte. 

Generally, the metal being analysed isn't present in its pure elemental form, so the analyte must be broken down (a step called digestion) in a series of standards (in liquid form). Standards are used to construct calibration curve. Each standard passes through a nebulizing chamber, where oxygen is injected in the liquid sample, resulting in very fine spray. The spray then enters an atomizer (generally a flame atomizer/graphite furnace/electrically heated), where the sample becomes atomized. The hollow cathode lamp irradiates the atomized sample, which passes through the monochromator. This is an optical component that transmits a beam of light with a very narrow range (basically a single colour), by reflecting away the unwanted wavelengths. The results are then amplified and the absorption spectrum is output to a computer display.

Modern AAS devices are computer-controlled, where the data is automatically recorded processed onto the computer. The AAS device is extremely sensitive - in the range of μg dm^-3 ! 

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As always, thanks for reading! 

Don't forget to check out my Patreon,  if you like the content I'm putting out:

 https://www.patreon.com/MrElement 

Links provided bring you to some of the info I used from the web. the first/second-year university textbooks:

• "Inorganic Chemistry," 4ED, by Housecroft & Sharpe, Pearson Ed. Ltd., 'Chapter 4 - Experimental techniques,' pgs 90-98. You can buy it here.
• "Fundamentals of Analytical Chemistry," 9ED, Skoog & West, Pearson Ed. Ltd., 'Chapter 28 - Atomic Spectroscopy,' pgs 774-775, 790-799. You can buy it here.

Chemical Wonders: Experimental Techniques (Part 1) - Separation and Purification: Chromatography and Recrystallization

- Reposting from my blog on minds.com - 

Identifying and analysing compounds is an important part of chemistry, the modern laboratory provides various analytical techniques. These methods and techniques can be roughly grouped depending on their usage:

• in determining molecular structure;
• in investigating the bonds of the analyte, how and which atoms are connected in the molecule; and what the oxidation states of each element in the analyte;
• in determining molecular formulae and analysing the composition of the analyte.

Separating and purifying compounds

Once you have made your compound and before you begin to analyse the structure and molecular formula, you must first separate your product and purify it. It is essential to separate and purify your analyte from other products in your reaction mixture because you would get an inaccurate reading.

Gas Chromatography

Gas chromatography (GC) involves very small amounts of gas, and is typically used to separate particularly volatile components from a reaction mixture. GC depends entirely on the various interactions between the components in the mobile phase (a carrier gas such as Helium, He and the sample gas) and the stationary phase containers (silica,SiO₂; or alumina, Al₂O₃). Typically, GC is combined with mass spectrometry (MS) in a machine called a GC-MS instrument, so that the sample is separated, purified and an MS reading is made at once. 

The small sample of gas is injected directly into the coiled-shaped chromatography column, which is generally a capillary or microbore column. The temperature of the column can vary depending on the gas, from 50 °C to 250 °C. The gas may separate in any number of ways that aren't necessarily permanent:

• the gas could condense in the stationary phase;
• on the other hand, the compound could remain in the gas phase;
• the gas could even dissolve on the liquid surface present on the surface of the stationary phase.

The time it takes for these compounds to be eluted (washing the substance with a solvent present in the stationary phase. In this case, 'washing' the sample gas from the carrier gas) from the chromatography column is called the retention time. The retention time of a substance depends on various factors, e.g.:

• If the sample's components do not interact very well with the chromatography column, the retention time will be short. This is usually the case when the chromatography oven is heated at high temperatures, exciting the sample molecules so that they evaporate readily; OR because they are moving so rapidly they aren't able to interact with the stationary phase.
• Depending on the boiling point of the sample; for example, a sample with a higher boiling point than the liquid in the stationary phase will spend more time in the liquid state at the beginning of the coil. 
•  If the sample is more strongly absorbed by the column. In other words, the the solubility of the gas will determine how long the gas is carried by the carrier gas; a higher solubility means a shorter time in the gaseous phase and a higher retention time.

 Liquid Chromatography (LC)

Column liquid chromatography is a common method of purifying a product after synthesising it. This separation technique involves the mobile phase being a liquid, while the stationary phase is absorbed into the chromatography column or is stuck to a glass plate. There are many ways to do liquid chromatography, some examples are open-column or liquid chromatography combined with mass spectrometry (LC-MS).

The stationary phase of LC uses a crude material such as silica (SiO₂) or alumina (Al₂O₃), with a particle diameter of around 20 μm, which then absorbs the . Depending on the substance, the chromatography column is eluted after the sample has been absorbed. This allows different components of the reaction mixture to separate as they each have different solubilities. As such, the solvent that is used allows the sample to flow out of the column already separated from the reaction mixture. 

The retention factor (Rf) is the value that represents the ratio of the distance travelled down the column by the analyte to the solvent used. The solvent and the analyte species form an equilibrium; either the stationary and mobile phase are given preference, and can be represented by an equilibrium constant (K) and the activity (a):

K = a_stationary / a_mobile  

When the column is eluted with the solvent, different components of the reaction mixture are separated (some more easily than others), eventually separating the analyte in fractions. These fractions are collected as the exit the column. In open column chromatography, these fractions are usaually eluted using gravity or pressure ("flash LC"). Colored compounds are often detected using mass spectrometers or a UV spectrometer.

High-Performance Liquid Chromatography

 This particular type of chromatography is different from the usual LC, as induces high pressure (up to 40MPa) to the mobile phase and the stationary phase utilises very small particles, ranging from 3 to 10μm in diameter. The pump that induces the pressure alters the flow rate and is dependant on the analyte passing through the column. HPLC is often used to separate fullerenes, columns specifically designed for the separation of fullerenes are commercially available, such as Cosmosil^TM Buckyprep columns.

Once the sample enters the chromatography column and is eluted, fractions are monitored using a type of detector (such as UV-VIS, IR, MS, fluorescence etc.). Data in HPLC is recorded in terms of the absorbance, A, over the retention time.

Analytical machines typically have 3 to 25 cm long by 2-4 mm wide chromatography columns. There are two types of HPLC chromatography columns:

• Reversed-phase: Where the surface of the column is hydrophobic and combined with a polar solvent, usually aqueous MeOH, MeCN or tetrahydrofuran (THF). The sample fractions are then eluted based on decreasing polarity. 
• Normal-Phase: Uses solvents that have low polarity or are non-polar, and fractions are eluted based on increasing polarity; non-polar fractions elute first. The stationary phase comprises of a polar substance such as silica.

Recrystallization

 When the product of a reaction is a solid, it needs to be separated from the solution it formed in. This is normally the final stage of purification, separating the analyte from minor impurities, such as water.

• To begin, you need to know what your solid easily dissolves in at relatively high temperatures but not in low temperatures. Generally the rule is "like dissolves like," so for instance: polar and ionic/polar solids are soluble in polar solvents; while non-polar solids do not (they are soluble in non-polar solvents). The ideal solvent in one in which the impurities do not dissolve in.
•   The impure sample mixed with the solvent is heated and then filtered to remove the impurities.
• When the filtered solution cools down, it becomes saturated and the sample begins to crystallize as its solubility decreases. Rapid recrystallization produces very fine crystals, the longer it takes to cool, the larger the crystals become (generally). The purpose of this process is to minimize the impurities present in the crystal lattice: a purer the lattice leads to a more ordered (and well defined shape) crystal lattice. 
• After the crystals form, you filter the solution containing the crystals with a vacuum, a buchner flask is an example of a tool used for this purpose (see below). 

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The next section will focus on the analysis of the elements present in a compound, which allows us to predict the chemical formula of the analyte in question. 

Don't forget to check out my Patreon,  if you like the content I'm putting out:

 https://www.patreon.com/MrElement 

Links provided bring you to some of the info I used from the web. the second-year university textbook, "Inorganic Chemistry," 4ED, by Housecroft & Sharpe, Pearson Ed. LTD, Chapter 4, was used as a guide to write this post. You can buy it here.

Sowing the seeds of the future - What GMOs really are and why you shouldn't be afraid.

The Genetics of Life

As a species, we've domesticated and selectively bred animals and plants for our own benefit, and so have allowed those organisms to survive; we've changed the earth, for better or for worse, and have had a hand in breeding crops that have ensured our survival. One of the best examples of this is the wheat we find in our bread and pasta, so how did they come about?

Before I continue, I must first define a few things, because of the importance of genetics in the long history wheat in our past and present:

• Every living thing on earth has a genetic code that defines characteristics at the species level and of the individual. For example, in humans: eye colour and sex are determined by specific genetic sequences.
• These genetic sequences are found on chromosomes, which are more easily seen during cell division. Chromosomes are tightly wound bits of DNA that you inherit from your parents, and the amount you have is called ploidy. For example, (most) humans have 23 chromosomes that are paired, one set of 23 from mother and father, totalling 46 chromosomes. Therefore we are "diploid", displayed like this: 2n=2x=46.
• Polyploidy refers to cells and organisms that contain more than two paired chromosomes, inherited from the parents. Sometimes one species may breed with another the offspring is called an allopolyploid.
• Mutations are random changes in the DNA sequence, due to many factors. I won't go to specific but these can result in characters that are lethal, beneficial or have no effect on the individual's ability to survive.

The History of Bread and Pasta Wheat

We must now cast our eyes to the Near East, ~9500 years ago where a mutation occurred in a wild Einkorn wheat (Triticum monococcum ssp. aegilopoides) giving rise to what is now referred to as Einkorn wheat (Triticum monococcum ssp. monococcum, 2n=2x=14). Domesticated Einkorn wheat possesses a mutation that suppresses shattering, making it easier to harvest the wheat. The cultivation of Einkorn wheat dates back to ~9400 years ago and is now used as animal feed, primarily in Italy, Spain and Turkey the mountainous areas as an animal feed.

A precursor to the bread and pasta wheats, Triticum turgidum spp. diococcoides (2n=4x=28, AABB) arose as an allopolyploid when a while species of wheat (Triticum uratu, 2n=2x=14) and a closely related wild grass (Aegilopes speltoides; 2n=2x=14) bred to form the ancestor to the wheats we know and love.

Around 9000 years ago in the Fertile Crescent, a new wheat arose, called Emmer wheat (Triticum turgidum ssp. dicoccum; 2n=4x=28), which is a hulled wheat (the grains have no covering and exposes the whole seed) with much larger seeds than T. turgidum spp. dicoccoides. Emmer wheat was merely a stepping stone: about 7000 years ago, a mutation arose that allowed the grain to be free-threshing and a new species came about, called Durum wheat (T. turgidum ssp. durum) which is the wheat used for pasta. 

Bread wheat (Triticum aestivum, 2n=6x=42; AABBDD) arose ~6000 years ago in the Fertile Crescent, and as fate would have it, came about when domesticated Emmer wheat and a wild relative, Triticum tauschii (2n=4x=14) bred to form one of the most important grains in human history. 

Modern Farming and GMOs

Throughout this time period and to now, wheat growers have gone through major phases of selecting individuals for breeding (which can be considered a way of making GMOs) :

1. Subconscious and deliberate selection by wheat growers throughout history. These types of selection resulted in many characteristics being selected, such as choosing individuals with hardier spikes, those with fast and synchronous germination and plants which grow in a more erect habit. Both types of efforts enabled farmers to have an increased crop yield, better flour quality and larger grain size.
2. Scientifically planned breeding by scientists that specialise in genetics and agriculture, who then select individuals based on their lineage and most desirable characters, based on genetic evidence. And may lead to the use of breeding genetically engineered organisms with an array of better characteristics.

The most controversial, method of the modern farmer is genetic engineering - a process that may take from 6-15+ years of hard work and trial and error. These projects must go under rigorous testing, certification and regulatory approval by government agencies, for safety of consumption etc. The end result of this is plants that are more hardy, more resistant to herbicides and contain natural insect repellents, thus allowing farmers to produce more, and have bigger grains than their ancestors. Perhaps the most successful use of modern techniques was the Green Revolution in the 1960s, which allowed many countries to become self-sufficient when growing the hardy semi-dwarf wheats (albeit at a social cost). 

A livelier use of genetic engineering is perhaps the Gold Rice Project, which has allowed communities where Vitamin A deficiency is a very real threat to almost disappear, saving many lives and ensuring those communities do not suffer from it again. And of course, we have the "Super Bananas," which has the same goal of Golden rice, to help those communities with Vitamin A deficiency. Genetically engineered crops have the potential to do more good than they are currently doing so, if the uninformed stigma surrounding them were to disappear. 

. . .

The journey of one of the most important crop plants in human history is a beautiful one, and has arguably had the most influence in our agricultural habits, which evolved along with our way of making new foods from the crops in our back yards. It's important to remember that, the next time you enjoy pasta at your favourite restaurant, munch on a sandwich for lunch or have doubts on whether or not the bread your eating is "natural" enough.