Definitions

ADAMS

The Anti-Doping Administration & Management System (ADAMS) is a web-based database management system that provides a mechanism to assist stakeholders and athletes with coordinating anti-doping activities worldwide under the World Anti-Doping Code.

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Analyte

The target species of interest in the mixture being analyzed. There may be several analytes present in a single experiment, but in chromatography only one at a time will emerge from the column if the experiment has been set up correctly. Oftentimes problems result from different analytes that have similar retention times, because these need to be resolved by the column well enough to see each peak separately and not as a lumped together peak.

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Atmospheric pressure chemical ionization (APCI)

This technique is most often used in LCMS, and is a more gentle method of ionizing chemicals. In this technique, which is similar to electrospray up to a point, the analyte stream is sprayed through a nozzle and comes into contact with an electrode undergoing corona discharge. Depending on the mode of operation, negative or positive, the analyte is made into an ion by proton abstraction or proton addition by a reagent gas (methane, isobutane, ammonia, etc) This technique, like ESI, occurs with spraying and at atmospheric pressure, but it differs in how the charge is put on the analyte. This method works for molecules upto 1500 Da in weight, and generates singly charged ions.

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Biomarker

A substance, that can be measured and evaluated, the presence of which serves as an indicator of biological phenomena related to one or more of normal function, pathology, and/or pharmaceutical intervention.

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Batch operation

A batch operation means that the separation or process is carried out upon several separate discrete packages of input material called batches. Much like baking goods for a bake sale, first the plain cookies must be baked in a batch in the oven, taken out, and replaced by the perhaps the chocolate batch of cookies. This style of process is contrasted with continuous operation, where material is not processed in discrete packages with stops before and after the discrete package is changed, but as an unbroken stream. Commercial donut bakeries are a good contrast: dough is constantly being cut and formed into donuts, those are placed on a conveyor belt, they fry and are removed and packaged continuously, as already fried donuts get packaged, raw dough is dumped into the oil with no breaks in the continuous chain of supply.

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Bandshape

In chromatography, the relationship between concentration of an analyte as it exits the column and time is called the band shape. Bands should be gaussian ideally, but this is only true when the partition coefficient is independent of concentration. In reality the bandshape is often asymmetric. In an overloaded column, where there is too much solute, the stationary phase begins to act like solute instead of stationary phase, so the peaks skew right because the concentration gradually ramps up, saturates, then drops off sharply. The opposite problem is left skewed peaks, which are caused when small quantities of solute bind more strongly than large quantities to the stationary phase.

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Boiling point

The temperature at which a substance begins to change between liquid and vapor. The boiling point is defined as the point where the vapour pressure of the substance equals the atmospheric pressure. Mixtures may not have an easy to predict boiling point, which might be closer to a range than a point in actuality.

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Chiral chromatography

Chiral chromatography involves the use of a chromatographic stationary phase which is chiral in order to separate enantiomers. This is typically done using HPLC with a chiral column.

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Chromatography

“Chromo-” : color, “-Graphy”: writing; the term for a separations process in which a moving mixture of analyte and impurities (see mobile phase) is passed over or through an immobile solid chemical (see stationary phase) that specifically slows down different components of the mixture, separating those components on the basis of some microscopic interaction. The name is derived from the history, as the technique was first used by Mikhail Tsvet in the 1900s to separate the color components of simple pigments. Chromatography can either be used analytically to determine the amount of each component in the mixture or preparatively to purify large amounts of compounds, and these two goals are not mutually exclusive. Simple chromatography practiced in almost every undergraduate lab involves what is known as TLC (see thin layer chromatography). Chromatography generally has two modes: one in which the mobile phase is high polarity and the stationary phase is low polarity (see reversed phase chromatography), or vice versa (see normal phase chromatography), and these modes have different uses. Furthermore, chromatography can be performed either in the gaseous state (see gas chromatography) or in the liquid state (see liquid chromatography), and these two states have their own specific challenges. Chromatography separations are usually carried out on the basis of polarity interactions, usually called “affinity chromatography”, but they can also occur based on size (hydrodynamic volume) exclusion if the stationary phase has controlled pore size, they can even occur based on ion interactions if that is desired, they can occur based on surface adsorption, or even liquid partitioning if the liquid is bonded to a solid stationary phase thusly making a stationary liquid.

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CLIA

The Clinical Laboratory Improvement Amendments (1988) are the federal standards applicable to all U.S. laboratories that handle, evaluate, and test human specimens for diagnostic, preventative, and/or treatment purposes.

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Column

In chromatography the physical apparatus that the separation is carried out in, is called the column. It is usually named because in simple chromatography experiments the apparatus is indeed a tubular glass column. Columns come in two varieties generally: open tubular and packed. Open tubular columns should be thin and have a very thin coating of stationary phase to have fast transfer between phases. They have higher resolution, shorter experiment time, and increased sensitivity, but a lower sample capacity. They are also more efficient because they have increased linear flow rate and are longer, so the height of a plate is lower.  The vast majority of columns in GC are open tubular. Open tubular columns are more suitable for analytical work. For preparation however, the column of choice is the packed column, because it can separate much larger quantities of chemicals.

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Continuous operation

A continuous operation means that the process is carried out upon a monolithic, never interrupted input stream. Water treatment is a good example: impure water comes into the plant as one stream, it is processed in stages in a large serpentine tank, and by the time it flows to the end it is pure and ready to be used. The operation never needs to be stopped and restarted to reload water except for periodic maintenance, it just flows in and comes out ready. This is contrasted with batch operations, in which the feed needs to be replaced with new feed and the process needs to be restarted from the beginning each time.

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Derivatization

To change an analyte by adding or removing certain groups. In GCMS derivatization is often needed because the analyte may not be volatile enough to be put on the column, also it may be too polar for the available columns. The most basic chemical used in derivatization is MSTFA (N-methyl-N-(trimethylsilyl) trifluoroacetamide) or chemicals like it. What makes MSTFA special is that it generates the trimethylsilyl cation, which can replace protons in certain functional groups, and this cation makes the molecule more volatile and less polar.

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Diffusion

Transfer of mass from high concentration areas to low concentration areas. This is a physical process that occurs normally in any mixture due to thermal energy, though it can be greatly sped up by agitation or other methods of adding energy to a system. In chromatography diffusion is a major contributor to the quality of separation achieved on a column. It shows up 3 times in the Van Deemter eqn, as longitudinal diffusion down the axis of the column, and as diffusion between mobile and stationary phases. The parameter that measures the rate of diffusion is the diffusion coefficient, given in units of mol/(m^2*sec)

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Electron impact ionization (EI)

This ionization technique is most often used with GCMS, and it is probably the harshest ionization method possible. In this method, the sample is vaporized and put into a contact with a filament undergoing thermionic emission, and the sample is thusly bombarded with electrons. These electrons cause structural breakdown unique to each species, so when the fragments of the parent species enter the MS they show up as many different peaks with characteristic patterns. This makes EI the best ionization method for providing a “fingerprint” of a molecule. However, in certain applications a cleaner spectrum with only the main molecular ion and a few adducts is sufficient, so EI is not the best tool for every procedure. Note: this technique is often used in simple cases and where the molecular weight is below 600 Da, also note that the breakdown products must be thermally stable in the path of the GC for this to be useful.

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Electrospray ionization (ESI)

This technique can be paired with either GC or LC and is one of the more gentle ionization methods. The method involves spraying the analyte mixture through a charged nozzle, and when the voltage is high enough this spray will form a special shape with a jet stream on the end of it (see Taylor Cone). Because the liquid is charged, as the spray droplets get ejected from the jet stream the droplets collapse and ions are ejected. There are two theoretical mechanisms for how these ions get ejected from the droplet and move onto the detector, but those details are not important to understand the overall process. This technique works on a large size range of molecules, even proteins, because it produces multiply charged species thus extending the mass range of the detector.

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FDA

Food and Drug Administration, a United States executive branch agency under the federal department of health that is formally charged with making sure certain food products and drugs sold within the US are safe to consume. The FDA was initially given authority through the 1906 Pure Food and Drug Act. This act was passed in response to Upton Sinclair’s novel, The Jungle, in which various meat industry practices were exposed and shown to be dangerous and unsanitary. Even though the FDA does not inspect meat today, the act passed in 1906 mandated that labels with active ingredients be placed on drugs, and that food products had to have standards of production. The FDA is now a major regulatory agency that essentially controls the approval of all drugs that go to market. Complying with FDA regulations on safety and production is a major source of expenditure of money and time for pharmaceutical companies. These stringent regulations are in place to prevent disasters resulting from untested or not well studied medicine being used on the population. The FDA has control over many things that aren’t strictly food or drugs in a traditional sense. They also regulate tobacco products, condoms, radiation emitting electonics, and biotechnological devices such as insulin pumps.

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Gas chromatography

This is a separation technique for analytes in the gas phase, or analytes that are quite volatile. Oftentimes the analyte is not in the gas phase but because GC is more commonly available and cheaper, one will derivatize the analyte with a chemical to make it more volatile and less polar and compatible with the column. GC features very long thin capillary like columns coated with space age polymers and silica, and this is the stationary phase. The mobile phase is the analyte diluted with carrier gas, usually helium although nitrogen can be used in a pinch and hydrogen is used to effect a better separation. Modern GC machines can run on a single cylinder of helium for 15 years. Sample preparation can be easy for GC, or it could be more difficult if complex derivatization is required. Injection of sample is much more difficult than for LC because there is a split valve which controls how dilute the injected stream is, and the split ratio must be controlled to avoid overloading the column. GC can be optimized for time by programming a temperature gradient into the experiment. As the temperature increases, the vapour pressures of all components increase nonspecifically and all compounds are retained less on the column. By having a low initial temperature early eluting analytes are separated easily, and by ramping up the temperature the later eluting analytes come out in an acceptable time frame. For analytes that cannot tolerate higher temperatures, pressure gradients can be used similarly. Higher pressure increases the flow rate of the mobile phase and therefore decreases retention time. Finally, GC tends to have hard ionization methods such as EI (see electron impact) although positive or negative chemical ionization is available and this is a softer method.

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Gradient elution

Gradient elution is the practice of slowly increasing or decreasing the polarity of the solvent during HPLC to control retention factors and optimize for time. Control over the polarity usually results from mixing the choice solvent with another solvent of higher or lower polarity as needed. For example, in HPLC water is typically mixed in with acetonitrile as the solvent. Changing the balance of these two solvents changes the overall polarity so that early eluting nonpolar compounds are separated easily and later eluting polar compounds are separated quickly. Gradient elution is often segmented, where several proportions of solvents are selected, linearly, to provide a smooth gradient to separate, EG 25 A:75 B, 30 A: 70 B, and so on. Of note: gradient need not only apply to polarity, often times a pH gradient can be chosen in some types of chromatography to effect the separation.

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Hb mass

Absolute hemoglobin (Hb) content of the body in grams.

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Hematocrit

The ratio of the volume of red blood cells to total blood volume

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Hydrophilic

“Water loving” – the tendency of a substance to dissolve in water. High polarity substances and charged substances will usually dissolve in water more easily than in hydrocarbons because water’s dipole and polarizability make it easy for that solvent to accommodate a variety of different molecular electric fields.

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Hydrophobic

“Water fearing” – the tendency of a substance to fail to dissolve in water. Low polarity chemicals like longer chain hydrocarbons are hydrophobic, because water has high polarity and like dissolves like.

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Isocratic elution

Isocratic elution is the practice of using solvent of only one composition during chromatography. The solvent stays at the same polarity throughout the process. This is often sufficient, but may not be where there are significantly higher retention factors of later eluting compounds, because these compounds would elute too slowly to be practical.

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Lipophobic

“Fat fearing” – the tendency of a substance to fail to dissolve in longer chain hydrocarbons (fats, oils, hexane, toluene). This is analogous to the term hydrophobic, which means “water fearing,” but for lipids (“lipo-”). Higher polarity chemicals are often lipophobic because longer chain hydrocarbons are less polar, and like dissolves like. Charged species are also lipophobic because they need polar solvent to stabilize their charge.

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Lipophilic

“Fat loving” – the tendency of a substance to dissolve in longer chain hydrocarbons (fats, oils, hexane, toluene). This is analogous to the term hydrophilic, which means “water loving,” but for lipids (“lipo-”). Low polarity compounds are lipophilic because like dissolves like and longer chain hydrocarbons are more nonpolar.

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Liquid liquid extraction

LLE, also known as partitioning, is the process of extracting a solute within one liquid with another liquid in which that solute is more soluble. This is a very common separations lab technique but presents difficulties on the large scale. LLE is a very simple way to prepare certain matrices (beverages mainly) for use in GCMS or LCMS. The process is basically that two solvents should be chosen with different polarities and densities, usually one organic solvent and one aqueous, or one aqueous with a polymer matrix and the other simply aqueous. The target analyte should be soluble in the less dense phase and less soluble in the more dense phase. The two solvents, one containing solute, are then poured into a separatory funnel, an upside down conical flask with a tap at the bottom, and they are allowed to sit for some time to allow solute to distribute into the less dense phase. Once the solute has been extracted sufficiently, the bottom phase is tapped off just by gravity, leaving the pure aqueous phase enriched in solute. Oftentimes it may not even be as complicated as this. For instance, in analyzing the caffeine content of soda, it is enough to fill a small vial with 1 mL organic solvent, and then put in some microliters of beverage with caffeine using a micropipette, and then withdrawing only a sample of the organic layer on the bottom of the vial. For other matrices like urine and blood, LLE can be very much more complex, and is usually not the best choice.

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Liquid chromatography (LC)/High performance (also known as High pressure) liquid chromatography (HPLC)

Liquid chromatography (LC) is separating liquid phase mixtures based on their interaction with a stationary phase. LC works on nonvolatile chemicals with weights between 50 and 50 kDa optimally. In LC sample injection is easier than for GC as gas dilution does not need to take place, but sample preparation is typically more involved for LC. This is because in order to separate liquid phases effectively pH needs to be monitored and buffered, polarity needs to be optimized such that early eluting analytes get good separation and later eluting analytes do not take too much time to wash down the column, and often times the organic solvent used in the process needs to be carefully recycled or disposed of. Solvents used in LC typically are water for its high polarity, and acetonitrile for its low polarity, though many other solvents can be used depending on what is ideal for the analyte. LC is typically more expensive to run but works on a larger variety of analytes to a higher precision. LC used to be inefficient on its own as the column pore size was relatively larger to allow liquid to flow through it easily, so a variant called HPLC was created where the HP either stands for high performance or high pressure. In this modification, the pore size is greatly reduced, meaning the analyte can take even more paths through the column and have more interactions, but the cost of this is that the liquid will not move through the column very quickly if at all, so it needs pressure to push it through. This development lead to greatly increased column efficiency and allowed HPLC to become the industry standard in analysis and separation. Additionally, LC columns tend to be packed with stationary phase rather than have it coated on the walls, columns are much thicker than GC columns, but much shorter. Just as in GC, LC can be optimized for run time, but the most common method of doing this is not temperature control as in GC but solvent control. When the polarity of the mobile phase is changed the selectivity of the column changes specifically, so earlier eluting analytes can be separated effectively and later eluting analytes can be eluted quickly. Finally, LC tends to have soft ionization methods such as APCI (see atmospheric pressure chemical ionization) or even better ESI (see electrospray ionization).

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Mean Cell Volume (also Mean Corpuscular Volume)

The average volume of a red blood cell.

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Reticulocyte

A new, not fully-formed red blood cell.

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Off score

The ratio of the percentage of reticulocytes over the volume of mature red blood cells.

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Van Deemter’s Equation

The equation is given as H = A + B/v + (Cs +Cm)*v, where H is the height portion of the column equivalent to one theoretical plate, A is the factor that controls for the multiple paths the solute can take down the column, B is the contribution that controls for diffusion down the axial length of the column, v is the linear flow velocity,  and Cs/Cm are parameters that encode for mass transfer between stationary and mobile phases. The equation states simply that if there is more diffusion down the column, between phases, or there are many paths the solute can take, the column will be less efficient because the height of one plate is relatively larger by all these factors. Also, since this equation relates height of a plate to flow rate, that means that the flow rate can be optimized for a given column. Finally, note that this is only one possible relation that accounts for the height of a plate. Many more factors than flow rate can be considered and different equations will result.

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Theoretical plate

A “plate” is not a physical object in a column necessarily, but a point at which the target analyte is in equilibrium among all the phases. In distillation these “plates” could be actual slotted trays where liquid is forced through, so the concept is analogously named in chromatography despite likely having no real plates. The idea is that the more equilibrium stages one has in a column, the more separation that column can achieve, for both distillation and for chromatography. The Van Deemter equation in chromatography relates linear flow rate down the column to the height of one theoretical plate.

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Solvent

A substance in a mixture that constitutes the bulk phase of the mixture and has properties that allow it to complex other substances within itself. Table salt dissolving in water is a good example: water is the solvent and its polar structure allows it to approach oppositely charged Na and Cl ions, and pull them off of their crystal lattice into a “cage” of water molecules so that you can no longer even see the solid salt as a macroscopic object. The process by which a substance mixes microscopically into a solvent, such as with table salt and water, is called dissolution or solvation

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Solute

A substance in a mixture that constitutes the smaller fraction of molecules, that are surrounded by the bulk solvent molecules on a microscopic scale. The solute sees very little of other solute molecules as it is effectively complexed by the solvent, at least in an ideal solution.

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Solution

A mixture of one or more solutes and solvents. There is a concept of an ideal solution, and while some sources may not be clear on this, the only practical definition of ideal solutions are basically those solutions which follow Raoult’s (high solute concentration) or Henry’s Law. (low solute concentration) Ideal solutions by Raoult’s Law are in actuality very rare because Delta H_mix must be very close to 0, and this only happens when the solute and solvent are so similar as to be considered identical. Ideal Henry’s Law solutions are more possible because as the solution gets more dilute each molecule of solvent sees less other molecules of solvents, and thus has less self and cross interaction.

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Mixture

A combination of different substances, differing in molecular structure, in one overall package. It could be something like melting iron, vanadium, chromium, nickel, and carbon together to make stainless steel, a common alloy (metal mixture). It could be something like sugar dissolving in water, where you can no longer see one component but it still exists on a microscopic scale within the water. Or, it could be something like dispersing vinegar into oil temporarily to form a vinaigrette. Mixtures could be homogeneous or heterogeneous. Homogeneous mixtures are those in which a random sample of the mixture has the same composition as any other random sample, EG if you dissolve the maximum amount of sugar in your hot tea and you drink from the top it will be just as sweet if you were to pipette a sample out of the bottom of the cup. Heterogeneous mixtures are those in which random samples will have different compositions, much like taking a fork into an untossed salad and getting slightly different numbers and kinds of greens, different numbers and kinds of toppings, and a different amount of dressing.

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Pure substances

A “pure” substance, is a substance that consists of 100% of one type of molecule. This is contrasted with a mixture, which is composed of more than one type of molecule.

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Stationary phase

The stationary phase, as the name would suggest, is the substance that stays in place/does not move during chromatography, upon which the target analyte interacts so that a separation may be achieved on the basis of that interaction. In simple TLC, the stationary phase is the silica plate. In GC, the stationary phase is the inner wall of the column coated in various space age polymers or silica in order to have various polarities. In LC the stationary phase is the column packing material, usually silica resin of some sort.

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Mobile phase

In chromatography the mobile phase is the substance that carries the analyte and all other undesired compounds through the column such that they may interact with the stationary phase. In GC the stationary phase is helium carrier gas, and in LC the mobile phase varies depending on the mode of operation. For normal phase, the mobile phase is nonpolar and becomes more polar as needed. For reversed phase operation, the mobile phase starts polar and becomes nonpolar.

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Taylor Cone

The Taylor Cone is a phenomena from electrospray systems, which is what happens as you increase the voltage past a certain point on an electrospray system, called the threshold voltage. The liquid being sprayed will start to deform its shape into a convex sided special cone, which will break into a jet stream, that disperses into many tiny droplets as we desire in electrospray. In fact, the Taylor cone is just one possible shape that has properties that make dynamic control easier. If you were to increase the voltage even more past the threshold voltage, other dispersion phenomena are possible, but they may not produce as good a droplet distribution as easily.

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Polarity

Polarity colloquially among chemists is actually a name for two possible related phenomena. The more common idea of polarity is the idea that when two elements are bonded and have different electronegativities, they will have a skewed electron distribution across the bond. One side may have higher electron density than another, and thusly it will produce an electric dipole. Polarity can refer to the idea that as these dipoles get stronger, molecules have more electrostatic interactions amongst themselves and will interact favourably with charged species. Polarity of a bond can be measured in the unit Debye, which is actually a unit for the dipole moment of the bond. There is another related concept of polarity though, more accurately referred to as “polarizability” which is intricately linked to polarity. Polarizability means the ability to distort the shape of an electron cloud around a bond in response to neighboring electric fields. These two things are linked because in order for something to be polar it really must have a dipole to produce a useful electric field, and also be polarizable so it can interact with other objects easily. Water is a good example, not only does it have a large dipole moment, but it has a large dielectric constant meaning that it will react to the electric field of ions easily and will accommodate and solvate them.

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Solid Phase Extraction

SPE is the process of exposing a liquid ,mobile phase enriched in impurities and analyte to a slug of stationary phase adsorbent material. This material can be many things, most often some type of polymer or resin with the correct polarity groups attached to it. It can be run in normal phase, where the analyte is polar and the impurities are nonpolar, so therefore the stationary phase material is polar and the solvents used are nonpolar. Or, it can be run in reverse phase, where the target analyte is nonpolar, so the stationary phase is nonpolar and the solvents are polar. Taking normal phase as an example, the SPE cartridge is wetted with nonpolar solvent, then water and buffer, then polar analyte solution. The analyte bonds to the polar stationary phase and nonpolar solvent is washed down the column to elute nonpolar impurities. Once the target is sufficiently cleaned of impurities, a polar solvent can be washed down the cartridge to elute the analyte. SPE is the method of choice for cleaning urine and blood matrices. There may be easier and cheaper methods if there is not a large variety or number of compounds to separate and none of them are too exotic.

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Relative volatility

Represented by the greek letter alpha, this ratio is either the ratio of vapour pressures of two substances being separated by distillation or the ratio of their Henry’s Law constants. The greater the relative volatility, the easier it is to separate two compounds by distillation. If alpha ~= 1, the separation will be difficult, but in general if alpha ~=1.5 it should go well provided all other considerations for distillation are satisfied.

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Partition coefficient

The ratio of solubility of a substance in a nonpolar solvent (usually 1-octanol) to the solubility of that substance in polar solvent (usually water). The log of this ratio is a measure of how hydrophobic/hydrophilic a substance is, as the nonpolar phase is always reported in the numerator and the polar phase is always reported in the denominator. If something is hydrophobic, it will have a large log P value, and conversely if it is hydrophilic it will have a small log P value. Note, this is not so simple in reality because the solute may be unionized in nonpolar solvent but ionized in the water, so a true look at the partition coefficient will add up all contributions from ionized and unionized sources.

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Selectivity (LLE)

Analogous to the relative volatility but for LLE instead of distillation, this number is defined as the partition coefficient of a solute Z in solvents A and B multiplied by the ratio of concentration of Z in A to the concentration of Z in B.

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Retention factor

In chromatography the retention factor is defined as the partition coefficient multiplied by the ratio of the volume of the stationary phase to the volume of the mobile phase. This number tells you how well the solute is retained on the column in a separation, higher retention factor is usually better however if it is above 10 it could be a waste to increase the retention factor any more. In fact, to reduce experiment times in GCMS, the retention factor is lowered nonspecifically by increasing the temperature, so a temperature gradient is often run in GCMS to make analysis times lower. This works well as long as the retention factor does not get too low, 10 is a good rule of thumb but in many situations gains can be made at an acceptable loss to peak separation below this number. Note that the retention factor does not tell you absolute information, only relative. Something with kr=10, is retained twice as long on the stationary phase as something with kr=5, and something with kr=0 is not retained at all. Also note that since everything spends the same amount of time in the mobile phase, the retention factor is really comparing times spent in the stationary phase. Also note that retention factor is related to partition coefficients thusly: k = K_p (Vstationaryphase/Vmobilephase)

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Phase ratio

Phase ratio is a common index in chromatography used to compare retention on columns with different film thickness and column diameter. Say a certain separation calls for a column with .5 um film thickness and .25 mm diameter, but this column is nowhere to be found. To substitute for this column effectively, one needs to find a column that will provide similar retention times, so to do this simply calculate the phase ratio with the formula beta = column diameter / (4*film thickness) and find a column with a similar phase ratio. In this case, the phase ratio is 125, so looking at a phase ratio table hints that a column with 1 um film thickness and .53 mm diameter would work about the same.

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Selectivity (chromatography)

The selectivity in chromatography is the ratio of retention factors of two compounds moving down the same column. If the selectivity is high, that means the compound represented by the numerator retention factor is preferentially bound to the column and the denominator compound will elute first.

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Resolution (chromatography)

The resolution in chromatography is defined as twice the difference in retention times of two compounds divided by the sum of the base widths of both compounds’ peaks. The higher the resolution, the greater the separation between peaks, and therefore the better separation of compounds physically. When compounds co-elute, it can be difficult to tell the substances apart and it may cloud analysis. Therefore it is often in the best interest of the instrumentalist to achieve as great a resolution possible in the given time constraints.

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Retention time

In chromatography, the retention time is the time the target analyte takes to elute from the column completely, the sum of the time it takes for the analyte to spend in the mobile phase and in the stationary phase. The longer the retention time the more the interaction with the column.

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Thin layer chromatography (TLC)

A simple chromatography setup where a plate coated in silica is marked near the bottom with a straight line, and the nonvolatile target solution to be analyzed is dropped onto the line in one very controlled, small drop. Capillary action will pull the liquid up the plate and the various components will interact with the plate to different extents. Those that do not interact as much will reach higher up on the plate and a gradient of bands can be clearly seen where the chemicals stop on their journey up the plate. This is a 2D version of column chromatography, and works very similarly.

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Normal phase chromatography

In normal phase separation, the column consists of a solid support made usually of silica or alumina resin, and a nonpolar solvent, used as the mobile phase. Hydrophilic, polar, molecules in the mobile phase bind to the resin support and thusly hydrophobic, nonpolar, molecules are eluted off the column first. The strength of binding is related directly to analyte polarity. To eject the hydrophilic molecules from their stationary phase binding, a polar solvent can be washed down the column and it will elute the hydrophilic molecules. Note that the analyte interacts directly with the solid phase in this technique.

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Reversed phase chromatography

Reversed phase chromatography is the opposite of normal phase in that a polar solvent is used as the mobile phase and the stationary phase is a hydrophobic alkyl chain bonded to silica. In this technique the analyte interacts with a solvated layer of hydrophobic molecules, and the hydrophobic analytes are retained on the column while the hydrophilic analytes are eluted. Washing the column with nonpolar solvent will unbind the remaining analyte and it will elute. An important note is that reversed phase has become very popular because it tolerates a wide range of analytes as it uses polar solvent. Also, reverse phase separation has better control of pH and solvent fraction due to the fact that the solvent is polar, allowing for more fine tuned control of separation variables.

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Matrix assisted laser desorption ionization (MALDI)

This technique is often paired with a TOF detector (see time of flight) instead of a quadrupole MS. In this technique an organic crystal matrix that absorbs UV (usually something based on sinapinic acid) is loaded with analyte and excess protons, and then irradiated with a UV laser. This laser desorps/ablates a small portion of the bulk matrix and this becomes ionized in contact with the excess protons. It is a very gentle method of ionization and is primarily used for large molecules, some proteins upto 60 kDa have been studied using this technique. One reason for the larger range of mass, is because the TOF detector is capable of a larger mass range than a quadrupole.

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Time of Flight Detector (TOF)

This detector, often paired with matrix assisted laser desorption ionization (see MALDI), is used in some systems in place of a quadrupole. TOF analyzers have several advantages over quadrupoles in that they have very little size restriction, and they can analyze many ions in parallel. However, they provide little identifying information other than the m/z ratio. This is because of how they work: different ions with different m/z values are accelerated in a uniform electric field, and depending on that m/z they will have different velocities. The time taken to reach the director is measured, and then the velocity and therefore the m/z value are back calculated from this. TOF analyzers have a high detection rate (100-10000 spectra per second), although it requires a much lower operating pressure of about 10^-7 Pa.

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Quadrupole Detector

Quadrupole detectors are the most common detector used in mass spectroscopy. They consist of 4 cylindrical or hyperbolic rods arranged parallel axially, each end positioned on the four corners of a diamond. It used to be thought that hyperbolic rods were necessary but recently it was found that cylindrical bars are cheaper to make and also work decently. The x plane rods carry an AC field and a light positive DC field, the y plane rods carry a light AC field of opposite phase and a stronger negative DC field. Light ions will oscillate in the AC field of the x plane, and if they are too light they will oscillate too hard and crash into the x poles – the x plane poles are high pass filters. Heavy ions on the other hand, are pushed down strongly by the negative DC field in the y plane, so they crash into the poles. Light ions are focussed by the opposite phase AC field, and enter the detector – the y plane is a low pass filter. Some combination of these two effects allow ions to travel the length of the detector and be selected by m/z value. Note that since this detector does not analyze a range of continuous masses it is not a true spectrometer, instead it selects and analyzes only one mass at a time. The pressure inside a quadrupole is around 10^-4 Pa. There are also modifications to the standard 4 rod design, which include a more complex 3 dimensional quadrupole made out of two rounded conical end caps and a toroidal central ring, and a linear trap version which creates a potential well in the center of the quadrupole to trap ions for longer. These designs benefit from lower detection limits, however because of the concentrated nature of charge in the 3D trap they have a limitation in the number of ions they can hold. Also these 3D quadrupoles trade spectral scanning rate (they scan less total m/z values than a plain quadrupole) for resolution.

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Ion trap detector

In an ion trap, or orbitrap, ions are generated by a gentle ionization method, and they are fed into a vacuum tube with a barrel shaped electrode. The ions orbit around this barrel, and the Fourier transform of their frequency space motions gives the time domain, which can be analyzed just as in a TOF spectrometer. These detectors can analyze single ions, and are compatible with fast injection techniques, and sometimes could be cheaper than quadrupole detectors depending on the application. However they have strict pressure requirements, so they may not be ideal for some applications, the pressure is the lowest, around 10^-8 Pa.

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Selected Ion Monitoring

This tool is a way to clean up busy spectra. This basically means that the spectrometer will only scan for a few m/z values, no more than 5 in one frame of time. If there are many compounds eluted down a column and there is only one target analyte, the m/z can be found and set to only view that one analyte, cleaning up the spectrum. This is related to but distinct from extracted ion monitoring where all m/z are scanned but only the target selection of m/z is displayed.

Volume flow rate

The 3D capacity of solvent running down a column given in units of mL/min.

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Linear flow rate

How much distance is traversed by the solvent going down the column per unit time, given in units of cm/min. This is a 1D version of volume flow rate, obtained by dividing the volume flow rate by the cross sectional area of the column.

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Guard column/Retention Gap

A guard column is a length of empty capillary in front of the main column, usually with a silanized wall. Modern column guards are disposable cartridges composed of the same packed material as the column being used. The guard column exists to capture nonvolatile substances that would contaminate the real column and reduce its performance. The guard column can be saturated and will lead to irregular peak shapes from a column that usually produces symmetric ones, and if this happens it should be cut down to remove the offending section. A retention gap is physically the same thing, but its purpose is to improve peak shapes in on column/splitless injection. If a large amount of solution is sprayed into the column, the retention gaps allows extra time for that solvent to evaporate before it hits the column, preventing overloading.

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Split injection

In GC the sample can be loaded onto the column in a few different ways. One of them is split injection, used where the analyte concentration is no more than .1% of the sample. The smallest amount of sample possible should be injected in less than 1 second at high temperature to provide for fast evaporation. In split injection the sample is injected into a hot chamber, mixed with carrier gas, and before it enters the column some is discarded and only a fraction goes in, determined by the split ratio. Higher split ratio means more is discarded before it enters the column. Note that in this technique lower boiling analytes may have too high a pressure and may back out of the injector, so a septum purge valve is added which flushes carrier gas back through the entry port to prevent gas bleeding out and to control excess sample.

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Splitless injection

Where the analyte  is less than .01% of the sample, splitless injection can be used. In this method sample is directly injected into a hot port with the split valve closed, leading to 80% of the sample hitting the column. The initial part of the column should be set to about 50 C below the boiling point of the sample, to allow solvent to collect at the beginning of the column. As solute continues to flood in, it collects in the solvent creating a very tight well shaped band. This is called solvent trapping and it improves peak shape. If this technique is not used, the bands would be limited by the column flow rate. Another similar idea is cold trapping, in which the initial column temperature is even less, about 150 C lower than the boiling point. Low boilers and solvent elute quickly, while high boiling compounds accumulate and upon rapid warming, elute all at once. A more extreme version of this for low boiling solvents is called cryogenic focusing, where temperatures below room temperature is used.

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On column injection

For quantitative work, on column injection is preferred. It is also preferred where the analyte decomposes above boiling point. In this method, the hot injection port is bypassed completely, and the solution is injected directly into the column. For small columns, special needles are needed.

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Spiking or Co-chromatography

In this qualitative technique, a sample suspected of a compound is analyzed, then spiked with more of that suspected compound. If the peak for the analyte grows, then there is a good chance that the suspected sample contains the analyte. Confirmation ions are necessary however, to further illuminate the identity of the sample.

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Qualifier ion

The peak in a mass spectrum that is characteristic of the main ion being analyzed. This peak could be the second largest peak, or it could be any other peak that has a definite abundance in each spectrum of the analyte, that is repeatable from spectrum to spectrum. This peak is used to confirm the identity of the main ion if it has the correct m/z.

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Quantitative ion

The peak in a mass spectrum that corresponds to the analyte. Also known as the target ion or main peak.

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Carbohydrate (or Saccharide)[colloq: sugar, starch]

A chemical containing only carbon, hydrogen, and oxygen. Most, but not all carbohydrates have an empirical formula of Cx(H2O)y, which is where they derive their name – “hydrate of carbon”. Some carbohydrates should be familiar already like glucose and fructose, common in nutritional discussion, or even more commonly, their combination sucrose (table sugar). Sucrose is a disaccharide: (a common chemistry trope is adding a number prefix to specify the number of chain units “di-” (two) “saccharide” (sugar)) glucose and fructose bonded to each other via glycosidic linkage (-O- bond between sugars). Carbohydrates do more than nutrition however, the D in DNA stands for deoxyribose, a sugar that does not follow the general empirical formula for carbohydrates, yet makes up the backbone structure of DNA. Sugars can chain together in chemicals other than DNA also, they are known as oligosaccharides, and if one has ever read an ingredient list, maltodextrin should be a familiar example.

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Polymer

A long macromolecule made like a chain of individual units, the molecule is called a polymer (poly: many, mer: unit) and the units are called monomers (mono: single). There are biological polymers (see Proteins) and there are synthetic analogs like nylon, polyester, and polyethene to name a few. Polymers have many uses from providing macroscopic structure in packaging and in people, to providing microscopic control in polymer liquid liquid extraction and in people’s biological functions.

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Protein

A protein is a biological polymer (see polymer) made out of amino acid monomers. Proteins have many functions in the body, when they catalyze reactions they are called enzymes (see enzyme), but in all other cases such as regulating DNA replication, transporting molecules, and providing stimulus response, they are just called proteins. Proteins are assembled based on the genetic code contained within DNA: the sequence of nucleotide bases is read off from the DNA in a systematic fashion, then a copy is moved to an organelle called the ribosome, and as the nucleotides are read they bind like a lock and key to a specific amino acid, which is then tacked on to a growing chain and eventually the protein is created piece by piece. Proteins in general are very large molecules that have a complex 3D shape, they are said to be folded. Primary structure refers to the amino acid sequence, secondary refers to some shape motifs seen in proteins like alpha helices and beta pleated sheets, tertiary refers to the overall folded shape of the protein, and quaternary refers to what happens when several smaller proteins make up one larger more complex whole. As a chemical technologist you will encounter proteins, and they present unique challenges to study with traditional techniques because they are so large.

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DNA

DNA stands for deoxyribonucleic acid, to break this down “deoxy” means lacking an oxygen atom, “ribo” means ribose, a 5 carbon sugar, and “nucleic acid” means that attached to the one oxygen deprived ribose backbone, with an extra phosphate group, are some molecules called nucleotide bases. There are four nucleotide bases in DNA, adenine, thymine, guanine, and cytosine, and these four chemicals arranged in a specific sequence make up the genetic coding of an organism that governs how all biological molecules are to be made within that life. To make one macromolecule of DNA, there are actually two strands that constitute it that wind around each other in a double helix with some nucleotide bases pairing up to provide the overall structure. Adenine pairs with thymine via hydrogen bonds, guanine pairs with cytosine similarly, and these interactions allow two strands of DNA to hydrogen bond together and form a 3D structure that can be fed into proteins and unwound to be read. The fact that there are four bases that pair up two and two means that when one single strand of DNA has a certain sequence, the opposite paired strand has a complementary sequence. This fact is exploited by nature when it creates temporary working copies of DNA, referred to as RNA, which is chemically distinct from DNA in that it has a different sugar backbone that isn’t as stable, and it also has a substitute nucleotide for thymine called uracil, which is just unmethylated thymine.

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Mass Spectrometry

MS is a form of spectroscopy (looking at atoms and molecules based on their response to applied fields) in which a distribution of ions is created from a sample, and the mass to charge ratio of those ions is determined. There are several detectors that can be used in MS to achieve this goal, by several methods, and also several different ways of generating ions. A spectrometer generates a “spectrum,” this word mathematically meaning a continuous distribution of numbers, but for general purposes means a pattern of lines that acts as a “fingerprint” of the chemical being analyzed. The spectrum generated is typically a graph of intensity of ions registered at the detector, vs their back calculated mass to charge ratio, thusly showing how many of a particular mass of ion exists in a sample. Often times, there are many chemicals in a sample that could sit at around the same mass to charge ratio and lead to ambiguous analysis, so the sample must be separated by a fast and strong method, usually GC or LC (See Gas chromatography and Liquid chromatography), although other techniques might be used.

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Molecule

The smallest unit chemists typically deal with are atoms, physicists deal with subatomic particles. A larger unit than atoms, that chemists primarily deal with, is called a molecule. A molecule is a group of atoms “bonded” together in some way. That word is quoted because the bonds could be of various types. For instance, there is ionic bonding, where charge is involved and atoms are bonded based on electrostatic interaction, there is covalent where electrons are shared between atoms, and there is also hydrogen bonding, where an electronically rich atom partially provides charge density to a  proton via pointing its lone pair at the proton.

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Tandem MS

Tandem mass spectrometers contain multiple ion sorting (i.e., mass filtering) modules connected in series. Tandem mass spectrometers and are often denoted as MS/MS or MSn. They are considered to now be the industry standard for sophisticated mass spectrometry analysis because of their substantial advantages over single MS instruments. An important and commonly-found example of a MS/MS system is an instrument which contains three quadrupoles. Compared to single MS machines which contain only one quadrupole, “triple quad” instruments have three quadrupoles that operate in tandem, with the first quadrupole (Q1) and the third quadrupole (Q3) operating as mass filters while the second quadrupole in the series (Q2) operates as a collision cell.

With single quadrupole, the most selective mode is Selected Ion Monitoring (SIM), wherein fixed DC and RF voltages are applied across the quadrupole, which ensures that only a single m/z from the sample can pass through the quadrupole and arrive at the detector. All other m/z are filtered out.

In the case of tandem mass spectrometry, multiple reaction monitoring (MRM) becomes is similarly used to heightened sensitivity and selectivity. In this case, Q1 acts as a mass filter to select for a single m/z of interest and filter out all other ions that are accelerated into the quadrupole once the sample is ionized in the source. So far, this is exactly what occurs in single quad machines. Next, however, instead of striking a detector, the m/z ion which passes through Q1 enters Q2 where it collides with a neutral collision gas, such as Argon,­ in a process called Collision Induced Dissociation (CID). This fragments the precursor ion into new product ions, which all travel into Q3. As in Q1, Q3 acts as a mass filter and allows only a single m/z ion to pass through to the detector while all others are filtered away. In this way, the MRM regime functions as a dual mass filter, which greatly improves the signal-to-noise (S/N) over single quad instruments.

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WADA (World Anti-Doping Agency)

Initiated by the International Olympic Committee, WADA coordinates and monitors the battle against drugs in sports.

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