Terminology LC

Absorption: The process where a chemical entity enter the bulk of a liquid, solid or gasphase. In chromatography the term usually signifies the process by which asolute partitions into a liquid-like stationary phase.Additive:A compound added to the mobile phase to improve the chromatographicanalysis.
Adjusted Retention Volume, V´R ( or Time, t´R ): The retention volume ( or time ) minus the Hold-up volume ( or time ). Note the difference between this and the retention volume ( or time )
Adsorbent: The packing used in adsorption chromatography.Adsorption:The process where a chemical entity is accumulated on a surface.
Adsorption Chromatography: Separation based on differences in adsorption of the components to the stationary phase surface.
Adsorption isotherm: A plot of the amount of solute per solid phase unit ( weight, volume, areaetc) as a function of its concentration in the bulk phase ( liquid or gasphase ).
Affinity Chromatography: Chromatographic separation based on a specific interaction between the analyte and a ligand bound to the stationary phase surface.
Agarose:A separation medium for the separation of biomolecules. It is a highmolecular weight polysaccharide.

Alumina: Porous aluminium oxide used as an adsorbent in chromatography .

Amphoteric ion-exchange phase: A stationary phase that has both positively and negatively charged ionicgroups bonded to it.

Analyte: The chemical entity to be analyzed. In chromatography the term solute isalso frequently used.

Anion exchange chromatography: The chromatographic process that is used to separate anions by using anionized positively charged stationary phase. Tetraalkylammonium ions are often used as anion exchange functional groups.
Baseline: The part of the chromatogram where the detector measures the mobile phase only.
Bed Volume : Synonymous to Column Volume
BET-method: A method for determining the surface area of a solid that was developed byBrunauer, Emmet and Teller. It uses the known size of the nitrogen moleculein combination with experimental data of adsorption-condensation ofnitrogen to the solid.
Bonded phase : A stationary phase which is covalently bonded to the support particles or the inside wall of a tube.
Breakthrough volume: When a solute is continuously pumped through a column, it will start toelute at a certain volume, this is the breakthrough volume.
Capacity factor : See retention factor. IUPAC discourage its usage.
Capillary column: Columns with an inner diameter less than 0.5 mm.
Capillary LC: Liquid chromatography performed by using a capillary column.

Cartridge column: A column type that has no endfittings and is held in a cartridge holder. Thecolumn comprises a tube and packing contained by frits in each end of thetube.

Cation exchange chromatography: The chromatographic process that is used to separate cations by using aionized negatively charged stationary phase. A sulfonic acid is an oftenused cation exchange functional group.

Channeling: Poor packing or erosion creates voids in the packed bed. Channeling occursbecause the mobile phase moves more rapidly in these these voids than inother parts of the bed.

Chemisorption: Adsorption, usually irreversible, accompanied by a chemical reaction withthe solid surface.Chiral stationary phase:Stationary phases that can separate enantiomers.

Chlorosilane: A reagent which is used to create siloxane bonds with silanol groups. Threetypes are used, monochlorosilanes;R1R2R3-Si-Cl, dichlorosilanes; R1R2-SiCl2and trichlorosilanes; R1-SiCl3 . Ri can have various structures but is oftenan alkyl group.
Chromatograph (noun): The instrument which is used to carry out a chromatographic separation
Co-ion: A ion of the same sign of charge as the ionic groups making up thestationary phase.
Column: The tube and the stationary phase through which the mobile phase flow.
Column back pressure: The difference in pressure between the column inlet and outlet.

Column chromatography: The form of chromatography which uses a column or tube to fix the stationaryphase.

Column plate number: See Plate number.
Column switching : Two or more columns connected by switching valves. Fractions from one columnare switched to a second column.
Column Volume : The volume of the empty column tube.
Competing base: A basic compound, often a small amine, added to the mobile phase with theintention to improve the peak shape of a basic solute.

Counterion: When the term is used in ion exchange chromatography it means the ions addedto the mobile phase with charge opposite to the ions bonded to thestationary phase.

Coverage: The concentration of bonded phase on the silica support, usually expressedin mol/m2 or weight %.

Cross-links: Bonds that connect one polymer chain to another. Cross-linking is importantfor resins because it governs its swelling and diffusion characteristics.Degassing:The removal of dissolved gas from the mobile phase.
Detector: An instrument that measures the change in composition of the eluent.
Displacement chromatography: A form of non linear chromatography where the migration of the solutes isdue to a displacement by an additive that strongly adsorbs to the stationaryphase.

Dynamic coating: Modification of the properties of the stationary phase surface by using anadditive in the mobile phase that adsorbs to the surface.

Effluent : The mobile phase that exits the column.
Eluate: The solute - mobile phase mixture which exits the column.

Eluent: Another word for the mobile phase.

Eluite: The eluted solute.

Elute: The use of elution chromatography.

Elution: The passing of mobile phase through the chromatographic bed to transportsolutes.
(Size) Exclusion Chromatography: Separation based mainly on differences in molecular size. Differences in shape and/or charge may also contribute to the separation.
Extracolumn effects: The effect on bandbroadening by all parts in a chromatographic system,except the column.
Extra-column Volume: The volumes of the injector, detector and connecting tubes. ( The term dead-volume is often used for this volume, IUPAC discourage this term.)
Fast protein LC (FPLC): HPLC of proteins, generally in glass columns with spherical microbeads andmoderate pressure.
Flow rate: The volume of the mobile phase that passes through the column per unit time.
Frontal chromatography : A chromatographic technique in which the sample is continously added to thecolumn inlet.
Fronting: Asymmetry of a peak such that its rear in a chromatogram is steeper than its front.

Gel filtration chromatography (GFC): Chromatographic separation according to molecular size usually performed inan aqueous mobile phase on soft gels such as polydextrans.

Gradient elution: The chromatographic technique by which a mobil phase gradient is used tomodulate the retention times. Usually the mobile phase composition changesso that its strength increases with time.

Graphitized carbon packing: A stationary phase consisting of pure graphitic carbon.

Guard column: A small column that protects the analytical column from contamination, it isplaced between the injector and the analytical column.

Heart cutting: A term used in preparative chromatography and column switching for thecollection of the center of a peak.

High performance liquid chromatography (HPLC): A term coined for the modern and instrumentally developed form of columnliquid chromatography. It is characterised by high flow rates and high backcolumn pressure.
Hold-up Volume, ( VM ) ( or Time ( tM ) ): The volume ( or corresponding time ) of mobile phase required to elute a component that does not interact with the stationary phase. I.e. the component is not retained by the stationary phase.
Hydrophobic Interaction Chromatography (HIC): A chromatographic technique which is primarly used to separate proteins. Thetechnique is characterised by a hydrophilic solid support with a lowcoverage of short carbon chains. The mobile phase is a buffered watersolution with a steep gradient of decreasing salt concentration.

IC: Abbreviation for ion chromatography

Imprinted phases: Stationary phases which are generated in the presence of a template moleculeso that a "footprint" of the molecule is created on the stationary phase.The imprinted phase has a strong selectivity for the template molecule.

Indirect detection: A detection technique where the solute is indirectly detected by measuringthe change in mobile phase composition at column outlet. A prerequisite forthis technique is that the adsorption isotherm of a component in the mobilephase depends on the concentration of the solute. E.g. a non-UV absorbingsolute is indirectly detected with an UV-detector by adding an UV absorbingcomponent to the mobile phase. If the adsorption isotherm of this componentdepends on the concentration of the solute, its variation in concentrationat the column outlet, caused by the elution of the solute, can be detectedwith an UV-detector.
(Sample) Injector: A device by which a sample is introduced into the mobile phase.
Inlet: The part of the column where the mobile phase and the solute enter.

In-line filter: A filter that is placed between the column and the injector and whichprevents particulate matter to damage the column.

Interparticle porosity (ee): The interparticle porosity is; ee = Ve/Vc where Ve is the interstitialvolume and Vc the total column volume.

Interstitial volume: In chromatography; the volume between the particles in a packed column.

Intraparticle porosity(ei): The fraction of the particle volume that is in pores; ei = Vi/Vparticlewhere Vi is the intraparticle volume and Vparticle the particle volume.

Intraparticle volume: The volume inside the pores of the particles.
Ion Exchange Chromatography: Separation based on differences in the distribution between the mobile phase and a charged stationary phase.
Ion chromatography (IC): A technique in which low concentrations of ionic solutes are determined byusing ion exchangers of low exchange capacity and mobile phase with lowionic strength.

Ion exclusion: The exclusion of co-ions from the surface layer. In chromatography the ionexclusion effect implicates that co-ions migrates faster through the columnthan a neutral molecule.

Ion moderated partitioning chromatography: A technique used for separating carbohydrates by using a strong cationexchanger.

Ion pair chromatography: A form of reversed phase chromatography in which a charged organic molecule,the ion pair reagent, is added to the mobile phase. The ion pair reagentadsorbs to the stationary phase surface and creates a charged surface layer.Ions of opposite charge are attracted to the charged surface layer and ionsof the same charge are repelled . The retention of ions is modulated bychanging the concentration of ion pair reagent in the mobile phase.
Isocratic chromatography: A chromatographic run with a constant mobile phase composition
Linear chromatography: Chromatography performed in the linear range of the adsorption isotherm forthe solute.

Linear velocity: The velocity of the mobile phase through the column expressed as m/s. It isestimated as the column length divided by the time it takes for anon-retained compound to pass the column

Liquid chromatography: Chromatography by using a liquid as mobile phase, usually performed in acolumn.
Mobile phase: The fluid that flows through the chromatographic column.
Mobile phase velocity : The linear velocity ( u ) of the mobile phase through the column. It is usually estimated from the time it takes for an unretained compound to pass through the column, tM, and the column length, L. u = L / tM
Open-Tubular Column : A column in which the stationary phase coates the inner wall. The column diameter is usually small, e.g. 0.1 mm.
Partition Chromatography : Separation based mainly on differences in solubilities between the mobile and stationary phase.
Packed Column : A column containing a solid packing material.
Peak: The part of the chromatogram where the detector response is caused by a solute.
Peak Area : The area of the peak as registered by the detector.
Peak maximum : The point on the peak where detector response is maximum.
Peak-Width : The width of the peak registrered by the detector. It may be represented in the dimension time or volume. For a Gaussian formed peak, the peak-width is related to the standard deviation (s) of the peak. The peak width can be estimated by several different methods. For example:
Peak-width at Base, wb = 4s
Peak-width at Half Height, wh = 2.355s
Peak-width at Inflection Point, wi = 2s
Phase ratio : A characteristic constant of a column. It is a measure of the volume ( or area ) of the stationary phase per unit volume of the mobile phase in the column.
Plate Height ( H) : The column length ( L ) divided by the plate number:
H = L / N
Plate Number ( N ) A dimensionless number that is a measure of the effectivity of a column.
N = ( VR / s)2
Pressure drop : The difference in pressure between the inlet and outlet in a chromatographic system
Reduced mobile phase velocity ( n ) : A dimensionless measure of the mobile phase velocity compared to diffusion into the pores. n = u*dp/DM
where u = linear velocity of the mobile phase; dp = particle diameter and DM = diffusion coefficient of the solute in the mobile phase.
(Peak ) Resolution ( Rs )
A measure how well two peaks are separated. It is defined as:
Rs = 2(tR2 - tR1) / (wb1 + wb2)
tR = Retention time, wb = peak width at base
Reduced Plate Height ( h ) : A dimensionless number defined as the ratio of the plate height divided by the particle diameter.
Relative retention time( usually denoted RRT) :RRT= tR2/tR1 = VR2/VR1
Note the difference between this and the separation factor. RRT is often used to identify peaks from system to system.
Retention Factor (k): The ratio of the adjusted retention volume ( or time ) and the hold-up volume ( or time ); k = V´R/ VM = t´R / tM
The retention factor has for many years also been called the capacity factor, k´. This usage is not recommended by IUPAC.
Retention Volume ( VR ) ( or Time ( tR ): The volume ( or corresponding time ) of mobile phase that passes through the column between sample injection and the emergence of the peak maximum. Note the difference between this and the adjusted retention volume ( or time ).
Separation Factor ( a ) : The relative retention values for two adjacent peaks;
a = V´R2/V´R1 = k2 / k1
V´R2 is chosen to be the larger value so that the separation factor becomes larger than unity.
Solid support : The solid that holds the stationary phase.
Solute : A term for the sample components.
Stationary phase : One of the two phases in a chromatographic system. In a chromatographic system the analyte is distributed between the mobile phase and the stationary phase.
Tailing : Asymmetry of a peak such that its front in a chromatogram is steeper than its rear.
Void Volume : The volume in the column that is filled with the mobile phase. In the ideal case it is equal to the mobile phase hold up volumne.

History of HPLC

Prior to the 1970's, few reliable chromatographic methods were commercially available to the laboratory scientist. During 1970's, most chemical separations were carried out using a variety of techniques including open-column chromatography, paper chromatography, and thin-layer chromatography. However, these chromatographic techniques were inadequate for quantification of compounds and resolution between similar compounds. During this time, pressure liquid chromatography began to be used to decrease flowthrough time, thus reducing purification times of compounds being isolated by column chromatogaphy. However, flow rates were inconsistant, and the question of whether it was better to have constant flow rate or constant pressure was debated. (Analytical Chem. vol 62, no. 19, oct 1 1990).
High pressure liquid chromatography was developed in the mid-1970's and quickly improved with the development of column packing materials and the additional convenience of on-line detectors. In the late 1970's, new methods including reverse phase liquid chromatography allowed for improved separation between very similar compounds.

By the 1980's HPLC was commonly used for the separation of chemical compounds. New techniques improved separation, identification, purification and quantification far above the previous techniques. Computers and automation added to the convenience of HPLC. Improvements in type of columns and thus reproducibility were made as such terms as micro-column, affinity columns, and Fast HPLC began to immerge.
The past decade has seen a vast undertaking in the development of the micro-columns, and other specialized columns. The dimensions of the typical HPLC column are: XXX mm in length with an internal diameter between 3-5 mm. The usual diameter of micro-columns, or capillary columns, ranges from 3 µm to 200 µm. Fast HPLC utilizes a column that is shorter than the typical column, with a length of about 3 mm long, and they are packed with smaller particles.
Currently, one has the option of considering over x# types of columns for the separation of compounds, as well as a variety of detectors to interface with the HPLC in order to get optimal analysis of the compound. We hope this review will provide a reference which all levels of HPLC users will be able to find quick answers to their HPLC problems.
Although HPLC is widely considered to be a technique mainly for biotechnological, biomedical, and biochemical research as well as for the pharmaceutical industry, these fields corrently comprise only about 50% of HPLC users.(Analytical Chem. vol 62, no. 19, oct 1 1990). Currently HPLC is used by a variety of fields including cosmetics, energy, food, and environmental industries.

HPLC - Mobile Phase

The mobile phase in HPLC refers to the solvent being continuously applied to the column, or stationary phase. The mobile phase acts as a carrier for the sample solution. A sample solution is injected into the mobile phase of an assay through the injector port. As a sample solution flows through a column with the mobile phase, the components of that solution migrate according to the non-covalent interactions of the compound with the column. The chemical interactions of the mobile phase and sample, with the column, determine the degree of migration and separation of components contained in the sample. For example, those samples which have stronger interactions with the mobile phase than with the stationary phase will elute from the column faster, and thus have a shorter retention time, while the reverse is also true. The mobile phase can be altered in order to manipulate the interactions of the sample and the stationary phase. There are several types of mobile phases, these include: Isocratic, gradient, and polytyptic.
In isocratic elution compounds are eluted using constant mobile phase composition. All compounds begin migration through the column at onset. However, each migrates at a different rate, resulting in faster or slower elution rate. This type of elution is both simple and inexpensive, but resolution of some compounds is questionable and elution may not be obtained in a reasonable amount of time.

In gradient elution different compounds are eluted by increasing the strength of the organic solvent. The sample is injected while a weaker mobile phase is being applied to the system. The strength of the mobile phase is later increased in increments by raising the organic solvent fraction, which subsequently results in elution of retained components. This is usually done in a stepwise or linear fashion.

Isocratic Vs. Gradient Elution
The Knox equation describes column efficiency or plate number N in relation to certain experimental conditions, such as column length, column diameter, temperature, flow-rate, molecular weight, etc.

Plate number N is equal to plate height value H divided by particle diameter (dp). Plate height value H is in turn equal to column length L divided by N. Two of the Knox coefficients, B and C, depend on k' and size of the compound. In the equations above, k' in the isocratic equations is replaced with average k' in the gradient equations. In fact, this is the only difference in the bandwidth and resolution equations between the two. Thus, separation and height of the peak are dictated by the exact same conditions for both isocratic and gradient elution (snyder -1983).

From the equation for capacity factor in gradient elution, it can be seen that average k' value depends on flow-rate, gradient time, and column dead volume. This differs in isocratic elution where k' is not dependent on time of separation, flow- rate, or column dimensions.
A special feature in gradient elution is linear-solvent strength (LSS) gradients. These give approximately equal values of average k' for samples eluting at different times during separation. This is the reason why gradient elution can yield constant bandwidths for different compounds and equal resolution for pairs of compounds which have similar alpha or separation factor values.
Polytyptic Mobile Phase, sometimes referred to as mixed-mode chromatography, is a versatile method in which several types of chromatographic techniques, or modes, can be employed using the same column. These columns contain rigid macroporous hydrophobic resins covalently bonded to a hydrophilic organic layer. SEC, IEC, hydrophobic or affinity chromatography are some of the methods that may be utilized. By changing the the mobile phase, the mode of separation is thereby changed which allows the chromatographer to achieve the desired selectivity in the separations.

HPLC - Stationary Phase

The stationary phase in HPLC refers to the solid support contained within the column over which the mobile phase continuously flows. The sample solution is injected into the mobile phase of the assay through the injector port. As the sample solution flows with the mobile phase through the stationary phase, the components of that solution will migrate according to the non-covalent interactions of the compounds with the stationary phase. The chemical interactions of the stationary phase and the sample with the mobile phase, determines the degree of migration and separation of the components contained in the sample. For example, those samples which have stronger interactions with the stationary phase than with the mobile phase will elute from the column less quickly, and thus have a longer retention time, while the reverse is also true. Columns containing various types of stationary phases are commercially available. Some of the more common stationary phases include: Liquid-Liquid, Liquid-Solid (Adsorption), Size Exclusion, Normal Phase, Reverse Phase, Ion Exchange, and Affinity.
Liquid-Solid operates on the basis of polarity. Compounds that possess functional groups cabable of strong hydrogen bonding will adhere more tightly to the stationary phase than less polar compoounds. Thus, less polar compounds will elute from the column faster than compounds that are highly polar.
Liquid-Liquid operates on the same basis as liquid-solid. However, this technique is better suited for samples of medium polarity that are soluble in weakly polar to polar organic solvents. The separation of non-electrolytes is achieved by matching the polarities of the sample and the stationary phase and using a mobile phase which possesses a markedly different polarity.
Size-Exclusion operates on the basis of the molecular size of compounds being analyzed. The stationary phase consists of porous beads. The larger compounds will be excluded from the interior of the bead and thus will elute first. The smaller compounds will be allowed to enter the beads and will elute according to their ability to exit from the same sized pores they were internalized through. The column can be either silica or non-silica based. However, there are some size-exclusion that are weakly anionic and slightly hydrophobic which give rise to non-ideal size-exclusion behavior.
Normal Phase operates on the basis of hydrophilicity and lipophilicity by using a polar stationary phase and a less polar mobile phase. Thus hydrophobic compounds elute more quickly than do hydrophilic compounds.
Reverse Phase operates on the basis of hydrophilicity and lipophilicity. The stationary phase consists of silica based packings with n-alkyl chains covalently bound. For example, C-8 signifies an octyl chain and C-18 an octadecyl ligand in the matrix. The more hydrophobic the matrix on each ligand, the greater is the tendancy of the column to retain hydrophobic moieties. Thus hydrophilic compounds elute more quickly than do hydrophobic compounds.
Ion-Exchange operates on the basis of selective exchange of ions in the sample with counterions in the stationary phase. IE is performed with columns containing charge-bearing functional groups attached to a polymer matrix. The functional ions are permanently bonded to the column and each has a counterion attached. The sample is retained by replacing the counterions of the stationary phase with its own ions. The sample is eluted from the column by changing the properties of the mobile phase do that the mobile phase will now displace the sample ions from the stationary phase, (ie. changing the pH).
Affinity operates by using immobilized biochemicals that have a specific affinity to the compound of interest. Separation occurs as the mobile phase and sample pass over the stationary phase. The sample compound or compounds of interest are retained as the rest of the impurities and mobile phase pass through. The compounds are then eluted by changing the mobile phase conditions.

HPLC Injectors

Samples are injected into the HPLC via an injection port (Rheodyne Type Injector). The injection port of an HPLC commonly consists of an injection valve and the sample loop.

The sample is typically dissolved in the mobile phase before injection into the sample loop. The sample is then drawn into a syringe and injected into the loop via the injection valve. A rotation of the valve rotor closes the valve and opens the loop in order to inject the sample into the stream of the mobile phase. Loop volumes can range between 10 µl to over 500 µl. In modern HPLC systems, the sample injection is typically automated.
Stopped-flow Injection is a method whereby the pump is turned off allowing the injecion port to attain atmospheric pressure. The syringe containing the sample is then injected into the valve in the usual manner, and the pump is turned on. For syringe type and reciprocation pumps, flow in the column can be brought to zero and rapidly resumed by diverting the mobile phase by means of a three-way valve placed in front of the injector. This method can be used up to very high pressures (willard - 1988).

HPLC Pumps

There are several types of pumps available for use with HPLC analysis, they are: Reciprocating Piston Pumps, Syringe Type Pumps, and Constant Pressure Pumps.
Reciprocating Piston Pumps consist of a small motor driven piston which moves rapidly back and forth in a hydraulic chamber that may vary from 35-400 µL in volume. On the back stroke, the separation column valve is closed, and the piston pulls in solvent from the mobile phase reservoir. On the forward stroke, the pump pushes solvent out to the column from the reservoir. A wide range of flow rates can be attained by altering the piston stroke volume during each cycle, or by altering the stroke frequency. Dual and triple head pumps consist of identical piston-chamber units which operate at 180 or 120 degrees out of phase. This type of pump system is significantly smoother because one pump is filling while the other is in the delivery cycle.
Syringe Type Pumps are most suitable for small bore columns because this pump delivers only a finite volume of mobile phase before it has to be refilled. These pumps have a volume between 250 to 500 mL. The pump operates by a motorized lead screw that delivers mobile phase to the column at a constant rate. The rate of solvent delivery is controlled by changing the voltage on the motor.
In Constant Pressure Pumps the mobile phase is driven through the column with the use of pressure from a gas cylinder. A low-pressure gas source is needed to generate high liquid pressures. The valving arrangement allows the rapid refill of the solvent chamber whose capacity is about 70 mL. This provides continuous mobile phase flow rates.

HPLC - Column

There are various columns that are secondary to the separating column or stationary phase. They are: Guard, Derivatizing, Capillary, Fast, and Preparatory Columns.

Guard Columns are placed anterior to the separating column. This serves as a protective factor that prolongs the life and usefulness of the separation column. They are dependable columns designed to filter or remove: 1) particles that clog the separation column; 2) compounds and ions that could ultimately cause "baseline drift", decreased resolution, decreased sensitivity, and create false peaks; 3) compounds that may cause precipitation upon contact with the stationary or mobile phase; and 4) compounds that might co-elute and cause extraneous peaks and interfere with detection and/or quantification. These columns must be changed on a regular basis in order to optimize their protective function. Size of the packing varies with the type of protection needed.
Derivatizing Columns- Pre- or post-primary column derivatization can be an important aspect of the sample analysis. Reducing or altering the parent compound to a chemically related daughter molecule or fragment elicits potentially tangible data which may complement other results or prior analysis. In few cases, the derivatization step can serve to cause data to become questionable, which is one reason why HPLC was advantageous over gas chromatography, or GC (brown-1990). Because GC requires volatile, thermally stabile, or nonpolar analytes, derivatization was usually required for those samples which did not contain these properties. Acetylation, silylation, or concentrated acid hydrolysis are a few derivatization techniques.
Capillary Columns- Advances in HPLC led to smaller analytical columns. Also known as microcolumns, capillary columns have a diameter much less than a millimeter and there are three types: open-tubular, partially packed, and tightly packed. They allow the user to work with nanoliter sample volumes, decreased flow rate, and decreased solvent volume usage which may lead to cost effectiveness. However, most conditions and instrumentation must be miniaturized, flow rate can be difficult to reproduce, gradient elution is not as efficient, and care must be taken when loading minute sample volumes.

Microbore and small-bore columns are also used for analytical and small volumes assays. A typical diameter for a small-bore column is 1-2 mm.

Like capillary columns, instruments must usually be modified to accommodate these smaller capacity columns (i.e., decreased flow rate). However, besides the advantage of smaller sample and mobile phase volume, there is a noted increase in mass sensitivity without significant loss in resolution (simpson -1987). --Capillary Electrophoresis

Fast Columns- One of the primary reasons for using these columns is to obtain improved sample throughput (amount of compound per unit time). For many columns, increasing the flow or migration rate through the stationary phase will adversely affect the resolution and separation. Therefore, fast columns are designed to decrease time of the chromatographic analysis without forsaking significant deviations in results. These columns have the same internal diameter but much shorter length than most other columns, and they are packed with smaller particles that are typically 3 µm in diameter. Advantages include increased sensitivity, decreased analysis time, decreased mobile phase usage, and increased reproducibility (DiCesare-1987).
Preparatory Columns- These columns are utilized when the objective is to prepare bulk (milligrams) of sample for laboratory preparatory applications. A preparatory column usually has a large column diameter which is designed to facilitate large volume injections into the HPLC system.

Accessories important to mention are the back-pressure regulator and the fraction collector. The back-pressure regulator is placed immediately posterior to the HPLC detector. It is designed to apply constant pressure to the detector outlet which prevents the formation of air bubbles within the system.

This, in turn, improves chromatographic baseline stability. It is usually devised to operate regardless of flow rate, mobile phase, or viscosity.The fraction collector is an automated device that collects uniform increments of the HPLC output.

Vials are placed in the carousel and the user programs the time interval in which the machine is to collect each fraction. Each vial contains mobile phase and sample fractions at the corresponding time of elution. Packings for columns are diverse since there are many modes of HPLC.

They are available in different sizes, diameters, pore sizes, or they can have special materials attached (such as an antigen or antibody for immunoaffinity chromatography).

Packings available range from those needed for specific applications (affinity, immunoaffinity, chiral, biological, etc.) to those for all-purpose applications.

The packings are attached to the internal column hull by resins or supports, which include oxides, polymers, carbon, hydroxyapatite beads, agarose, or silica, the most common type (brown-1990).

HPLC Detectors

The detector for an HPLC is the component that emits a response due to the eluting sample compound and subsequently signals a peak on the chromatogram. It is positioned immediately posterior to the stationary phase in order to detect the compounds as they elute from the column. The bandwidth and height of the peaks may usually be adjusted using the coarse and fine tuning controls, and the detection and sensitivity parameters may also be controlled (in most cases). There are many types of detectors that can be used with HPLC. Some of the more common detectors include: Refractive Index (RI), Ultra-Violet (UV), Fluorescent, Radiochemical, Electrochemical, Near-Infra Red (Near-IR), Mass Spectroscopy (MS), Nuclear Magnetic Resonance (NMR), and Light Scattering (LS).

Refractive Index (RI) detectors measure the ability of sample molecules to bend or refract light. This property for each molecule or compound is called its refractive index. For most RI detectors, light proceeds through a bi-modular flow-cell to a photodetector. One channel of the flow-cell directs the mobile phase passing through the column while the other directs only the mobile phase. Detection occurs when the light is bent due to samples eluting from the column, and this is read as a disparity between the two channels.
Ultra-Violet (UV) detectors measure the ability of a sample to absorb light. This can be accomplished at one or several wavelengths:
A)Fixed Wavelength measures at one wavelength, usually 254 nm
B)Variable Wavelength measures at one wavelength at a time, but can detect over a wide range of wavelenths
C)Diode Array measures a spectrum of wavelengths simulateneously.

UV detectors have a sensitivity to approximately 10-8 or 10 -9 gm/ml.

Fluorescent detectors measure the ability of a compound to absorb then re-emit light at given wavelengths. Each compound has a characteristic fluorescence. The excitation source passes through the flow-cell to a photodetector while a monochromator measures the emission wavelengths. Has sensitivity limit of 10-9 to 10-11 gm/ml.

Radiochemical detection involves the use of radiolabeled material, usually tritium (3H) or carbon-14 (14C). It operates by detection of fluorescence associated with beta-particle ionization, and it is most popular in metabolite research. Two detector types:
A)Homogeneous- Where addition of scintillation fluid to column effluent causes fluorescence.
B)Heterogeneous- Where lithium silicate and fluorescence caused by beta-particle emission interact with the detector cell.
Has sensitivity limit up to 10-9 to 10-10 gm/ml.

Electrochemical detectors measure compounds that undergo oxidation or reduction reactions. Usually accomplished by measuring gain or loss of electrons from migrating samples as they pass between electrodes at a given difference in electrical potential.
Has sensitivity of 10-12 to 10-13 gm/ml

Mass Spectroscopy (MS) Detectors- The sample compound or molecule is ionized, it is passed through a mass analyzer, and the ion current is detected. There are various methods for ionization:
A) Electron Impact (EI)- An electron current or beam created under high electric potential is used to ionize the sample migrating off the column.
B)Chemical Ionization- A less aggresive method which utilizes ionized gas to remove electrons from the compounds eluting from the column.
C)Fast Atom Bombarbment (FAB)- Xenon atoms are propelled at high speed in order to ionize the eluents from the column.
Has detection limit of 10-8 to 10-10 gm/ml.

Nuclear Magnetic Resonance (NMR) Detectors- Certain nuclei with odd- numbered masses, including H and 13C, spin about an axis in a random fashion. However, when placed between poles of a strong magnet, the spins are aligned either parallel or anti-parallel to the magnetic field, with the parallel orientation favored since it is slightly lower in energy. The nuclei are then irradiated with electromagnetic radiation which is absorbed and places the parallel nuclei into a higher energy state; consequently, they are now in "resonance" with the radiation. Each H or C will produce different spectra depending on their location and adjacent molecules, or elements in the compound, because all nuclei in molecules are surrounded by electron clouds which change the encompassing magnetic field and thereby alter the absorption frequency.

Light-Scattering (LS) Detectors- When a source emits a parallel beam of light which strikes particles in solution, some light is reflected, absorbed, transmitted, or scattered. Two forms of LS detection may be used to measure the two latter occurrences:
A) Nephelometry- This is defined as the measurement of light scattered by a particulate solution. This method enables the detection of the portion of light scattered at a multitude of angles. The sensitivity depends on the absence of background light or scatter since the detection occurs at a black or null background.
B) Turbidimetry- This is defined as the measure of the reduction of light transmitted due to particles in solution. It measures the light scatter as a decrease in the light that is transmitted through the particulate solution. Therefore, it quantifies the residual light transmitted. Sensitivity of this method depends on the sensitivity of the machine employed, which can range from a simple spectrophotometer to a sophisticated discrete analyzer. Thus, the measurement of a decrease in transmitted light from a large signal of transmitted light is limited to the photometric accuracy and limitations of the instrument employed.

Near-Infrared Detectors- Operates by scanning compounds in a spectrum from 700 to 1100 nm. Stretching and bending vibrations of particular chemical bonds in each molecule are detected at certain wavelengths. This is a fast growing method which offers several advantages: speed (sometimes less than 1 second), simplicity of preparation of sample, multiple analyses from single spectrum, and nonconsumption of the sample

What IS HPLC?

HPLC - High performance liquid chromatography is basically a highly improved form of column chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much faster.
It also allows you to use a very much smaller particle size for the column packing material which gives a much greater surface area for interactions between the stationary phase and the molecules flowing past it. This allows a much better separation of the components of the mixture.
The other major improvement over column chromatography concerns the detection methods which can be used. These methods are highly automated and extremely sensitive.

The column and the solvent
Confusingly, there are two variants in use in HPLC depending on the relative polarity of the solvent and the stationary phase.
Normal phase HPLC
This is essentially just the same as you will already have read about in thin layer chromatography or column chromatography. Although it is described as "normal", it isn't the most commonly used form of HPLC.
The column is filled with tiny silica particles, and the solvent is non-polar - hexane, for example. A typical column has an internal diameter of 4.6 mm (and may be less than that), and a length of 150 to 250 mm.
Polar compounds in the mixture being passed through the column will stick longer to the polar silica than non-polar compounds will. The non-polar ones will therefore pass more quickly through the column.
Reversed phase HPLC
In this case, the column size is the same, but the silica is modified to make it non-polar by attaching long hydrocarbon chains to its surface - typically with either 8 or 18 carbon atoms in them. A polar solvent is used - for example, a mixture of water and an alcohol such as methanol.
In this case, there will be a strong attraction between the polar solvent and polar molecules in the mixture being passed through the column. There won't be as much attraction between the hydrocarbon chains attached to the silica (the stationary phase) and the polar molecules in the solution. Polar molecules in the mixture will therefore spend most of their time moving with the solvent.
Non-polar compounds in the mixture will tend to form attractions with the hydrocarbon groups because of van der Waals dispersion forces. They will also be less soluble in the solvent because of the need to break hydrogen bonds as they squeeze in between the water or methanol molecules, for example. They therefore spend less time in solution in the solvent and this will slow them down on their way through the column.
That means that now it is the polar molecules that will travel through the column more quickly.
Reversed phase HPLC is the most commonly used form of HPLC.

Separation Ratio in GC

The separation ratio is simply the ratio of the solute distribution coefficients which depends only on the operating temperature and the chosen phase system. Most importantly, they are independent of both the mobile phase flow rate and the phase ratio of the column. Thus, the same separation ratio for two solutes would be obtained from either a packed column or a capillary column if the same temperature and the same phase system is used (at this time no exclusion effects from the support or stationary phase is assumed).
To identify a solute, a standard substance is added to the sample mixture and the separation ratio of the solutes of interest to the standard is measured. The ratios are then compared with those obtained for reference substances chromatographed under the same conditions. The ratios are calculated as the ratio of the distances in centimeters between the dead point and the maximum of each peak. If the flow rate is constant and data processing is employed, then the corresponding retention times can be used.

The effective plate number

The effective plate number is calculated in the same way as column efficiency, but uses the corrected retention distance, as opposed to the total retention distance in conjunction with the peak width.

As a consequence, the effective plate number is significantly smaller than the number of theoretical plates at low (k') values. The column efficiency and the effective plate number converge to the same value at high (k') values.

It follows, that the effective plate number more nearly corresponds to the actual resolving power of the column. Although the theoretical plate, as defined by the plate theory, has a practical significance and can be used in column design, the concept of the effective plate is not theoretically unsound and is related directly to the theoretical plate.

Detection and Quantitation Limits

Detection limit is the lowest concentration of analyte in a sample that can be detected, but not necessarily quantitated, under the stated experimental conditions.

Quantitation limit is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions.

Gas Chromatography Detectors

Gas chromatography detectors are devices that detect the presence of solute vapors as they are eluted from a gas chromatographic column.

Traces of vapor modify the properties of a gas far more extensively than traces of solute modify the properties of a liquid. As a consequence, the detection of vapors in gases is easier than the detection of traces of solutes in liquids.

It follows, that GC detectors are generally far more sensitive than LC detectors and there are more of them. The early GC detectors, the gas density balance (that responded to the change in density of the gas),

The thermal conductivity detector (TCD) (that responded to the change in specific heat and thermal conductivity of the gas) and the flame thermocouple detectors (that responded to the heat of combustion of a gas) all had sensitivities of about 5 x 10-7 g/ml at a signal to noise ratio of 2. As demands for improved sensitivity became greater to permit high efficiency columns to be operated, more sensitive GC detectors were developed.

One of the first high sensitive detectors to be described was the flame ionization detector (FID) (that responded to the ion current produced in the flame during the combustion of carbon containing solutes). This detector, although giving a relatively small ion current response (ionization efficiency only 0.0015%), had very low noise, and, consequently, a sensitivity of about 5 x 10-12 g/ml for n-heptane at a signal-noise ratio of 2.

A development of this detector, the nitrogen phosphorous detector (NPD) also provided very high sensitivities, but selectively to nitrogen and phosphorous containing compounds. The argon detector (developed at about the same time as the FID) provides sensitivities about an order of magnitude greater than the FID. It has an ionization efficiency of about 0.5 % but far greater noise than the FID. It functions by producing metastable argon atoms that have energies of 11.6 electron volts, sufficient to ionize almost all organic compounds. On collision with an organic molecule, the molecule is ionized and the metastable argon atom reverts to its normal state.

A further development of the argon detector was the electron capture detector (ECD) which exhibits a sensitivity nearly an order of magnitude greater than the argon detector, but is highly specific and will only give a significant response to electron capturing substances (e.g. halogenated compounds).

The group of GC detectors mentioned, represent those of historic interest and those most commonly used in general gas chromatography. There are many other GC detectors that are less common and that are generally used for very special applications.

How does the split/splitless injector work?

Packed column injectors consist of carrier gas inlet, a septum, septum purge, injector insert, heater block, and column connection; but the heart of this technological feat is another set of gas lines out of the injector-another path that the vaporized sample can take. This is called the split line or vent.

The manufacturers of these systems design them so that the carrier gas flow onto the column is constant-to maintain the chromatographic requirements of the column and yield reproducible retention times for analytes. At the same time, the amount of gas that goes out the split vent controls the amount of sample that enters the column.

If the split vent is closed, via a computer controlled split valve, then all of the sample introduced into the injector goes on the column.

If the split vent is open then most of the vaporized sample is thrown away to waste via the split vent and only a small portion of the sample is introduced to the column.

And finally, a very neat aspect of this is that the amount of gas exiting the split vent can be varied while keeping the flow onto the column constant.

This means that the AMOUNT of the split (called the split ratio) can be varied. A common split ratio is 50 to 1. That is, for every 50 units of gaseous sample that are thrown away to waste, 1 unit goes on the column.

The analyst keeps careful control of the split ratio so that results from the chromatography can still be quantified.

Chromatographic peaks that show up as, say 2.5 ng of compound X really represent 2.5 x 50 = 125 ng of analyte X in the original sample.

Also notice that this mass (125 nanograms) would have overloaded the column if all of it ended up on the capillary column. A split injection, and no sample dilution required

Height Equivalent to a Theoretical Plate

Since n depends on the length of the column, another parameter is used to express column efficiency. It is the height (length of column) equivalent to a theoretical plate, HETP, or just H
H=L/n
where L is the length of column in cm or mm. Thus H is the length of column which represents one theoretical plate in units of cm/plate or mm/plate.
The effect of flow on column efficiency is usually shown by plotting H versus flow rate or linear velocity. Such a plot is shown in the figure. Note that the H line goes through a minimum. The minimum occurs at the optimum flow velocity. The simplest equation for the curve in the H versus v figure is the van Deemter equation:
H=A+B/v+Cv
A van Deemter plot for gas chromatography can seen below. A, B and C are constants and v is the linear velocity, the carrier gas flow rate. The A term is independent of velocity and represents "eddy" mixing. It is smallest when the packed column particles are small and uniform.

The B term represents axial diffusion or the natural diffusion tendency of molecules. This effect is diminished at high flow rates and so this term is divided by v.

The C term is due to kinetic resistance to equilibrium in the separation process.

The kinetic resistance is the time lag involved in moving from the gas phase to the packing stationary phase and back again. The greater the flow of gas, the more a molecule on the packing tends to lag behind molecules in the mobile phase. Thus this term is proportional to v.

Theoretical Plates

The separation process in gas chromatography can be compared to a multiple distillation or a fractional distillation using a reflux column.

Gas chromatography uses relatively long packed or open tubular capillary columns and is subsequently far more efficient at separation than fractional distillations with short reflux columns. In addition, gas chromatography uses packing or stationary phases that can be liquid or solid and may exhibit an affinity toward the compounds being separated.

The column efficiency of a gas chromatography column is gauged by the number of theoretical plates, n.

The concept of a plate is a carry-over from the first fractionating columns which used discrete plates for separation. The chromatography column does not have discrete plates.

The number of theoretical plates is the number of discrete distillations that would have to be performed to obtain an equivalent separation. This number is commonly used as a measure of separation efficiency and is a useful number to use when comparing the performance of various chromatographic columns.

Gas chromatography columns normally have 1,000 to 1,000,000 theoretical plates as opposed to fractionating columns which normally operate in the range of 5-100 plates.
The number of theoretical plates, n, is a dimensionless number, which is related to the ratio between the retention time, tr, and the width of the peak containing the compound. If the peaks are reasonably symmetric, it can be assumed that they are Gaussian in shape. In this case, n is found from:
n=5.45(tr/W1/2)2.

The peak width at half height, W1/2, is found by drawing a line vertically from the peak maximum to the baseline, measuring half-way up the peak, drawing a horizontal line, and measuring the length of the horizontal line.

The retention time, tr, is measured at the point where the vertical line drawn through the maximum intersects the baseline. Both tr and W1/2 must be measured in the same units. Since the measurement is usually made from a recorder chart, the units are usually in cm, mm, or in.

n varies depending on the compound as well as the column packing material. So a column does not have a single n value. n also varies with the flow rate, and the column length. It is good practice to specify the column conditions and the compound used to determine n.

Gas Chromatography Columns

GC columns are of two designs: packed or capillary.

Packed columns are typically a glass or stainless steel coil (typically 1-5 m total length and 5 mm inner diameter) that is filled with the stationary phase, or a packing coated with the stationary phase.

Capillary columns are a thin fused-silica (purified silicate glass) capillary (typically 10-100 m in length and 250 µm inner diameter) that has the stationary phase coated on the inner surface. Capillary columns provide much higher separation efficiency than packed columns but are more easily overloaded by too much sample.

Stationary Phases
The most common stationary phases in gas-chromatography columns are polysiloxanes, which contain various substituent groups to change the polarity of the phase.

The nonpolar end of the spectrum is polydimethyl siloxane, which can be made more polar by increasing the percentage of phenyl groups on the polymer.

For very polar analytes, polyethylene glycol (a.k.a. carbowax) is commonly used as the stationary phase.

After the polymer coats the column wall or packing material, it is often cross-linked to increase the thermal stability of the stationary phase and prevent it from gradually bleeding out of the column.

What is Gas Chromatography

Gas Chromatography (GC) is used to separate volatile components of a mixture. A small amount of the sample to be analyzed is drawn up into a syringe.

The syringe needle is placed into a hot injector port of the gas chromatograph, and the sample is injected. The injector is set to a temperature higher than the components’ boiling points.

So, components of the mixture evaporate into the gas phase inside the injector. A carrier gas, such as helium, flows through the injector and pushes the gaseous components of the sample onto the GC column.

It is within the column that separation of the components takes place. Molecules partition between the carrier gas (the mobile phase) and the high boiling liquid (the stationary phase) within the GC column.

After components of the mixture move through the GC column, they reach a detector.

Ideally, components of the mixture will reach the detector at varying times due to differences in the partitioning between mobile and stationary phases. The detector sends a signal to the chart recorder which results in a peak on the chart paper. The area of the peak is proportional to the number of molecules generating the signal.

Commonly used GC detectors

1. Flame Ionization Detector (FID)

The detector is very sensitive towards organic molecules (10-12 g/s, linear range: 106 –107), but relative insensitive to a few small molecules e.g. N2, NOx, H2S, CO, CO2, H2O. If proper amounts of hydrogen/air are mixed, the combustion does not afford any ions. If other components are introduced that contain carbon atoms cations are produced in the effluent stream. The more carbon atoms are in the molecule, the more fragments are formed and the more sensitive the detector is for this compound (-- > response factor). However, due to the fact that the sample is burnt (pyrolysis), this technique is not suitable for preparative GC. In addition, several gases are usually required to operate a FID: hydrogen, oxygen (compressed air), and carrier gas.
2. Thermal Conductivity Detector (TCD)

This detector is less sensitive than the FID (10-5-10-6g/s, linear range: 103-104), but is well suited for preparative applications, because the sample is not destroyed. It is based on the comparison of two gas streams, one containing only the carrier gas, the other one the carrier gas and the compound. Naturally, a carrier gas with a high thermal conductivity e.g. helium or hydrogen is used in order to maximize the temperature difference (and therefore the difference in resistance) between two thin tungsten wires. The large surface-to-mass ratio permits a fast equilibration to a steady state. The temperature difference between the reference cell and the sample cell filaments is monitored by a Wheatstone bridge circuit.

3. Electron Capture Detector (ECD)

The detector consists of a cavity that contains two electrodes and a radiation source that emits b-radiation (e.g. 63Ni, 3H). The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions. If a compound is present that contains electronegative atoms, those electrons are “captured” and negative ions are formed, and the rate of electron collection decreases. The detector is extremely selective for compounds with atoms of high electron affinity (10-14 g/s), but has a relatively small linear range (~102-103). This detector is frequently used in the analysis of chlorinated compounds e.g. pesticides, polychlorinated biphenyls, which show are very high sensitivity

4. Mass Spectrometer (GC/MS)

Many GC instruments are coupled with a mass spectrometer, which is a very good combination. The GC separates the compounds from each other, while the mass spectrometer helps to identify them based on their fragmentation pattern.

Influences in GC separation

1. Polarity of the stationary phase

Polar compounds interact strongly with a polar stationary phase, hence have a longer retention time than non-polar columns. Chiral stationary phases based on amino acid derivatives, cyclodextrins, chiral silanes, etc are capable to separate enantiomers, because one form is slightly stronger bonded than the other one, often due to steric effects.

2. Temperature

The higher the temperature, the more of the compound is in the gas phase. It does interact less with the stationary phase, hence the retention time is shorter, but the quality of separation deteriorates.

3. Carrier gas flow

If the carrier gas flow is high, the molecules do not have a chance to interact with the stationary phase. So the quality of separation deteriorates.

4. Column length

The longer the column is the better the separation usually is. The trade-off is that the retention time increases proportionally to the column length. There is also a significant broadening of peaks observed, because of increased back diffusion inside the column.

5. Amount of material injected

If too much of the sample is injected, the peaks show a significant tailing, which causes a poorer separation. Most detectors are relatively sensitive and do not need a lot of material

6.Conclusion

High temperatures and high flow rates decrease the retention time, but also deteriorate the quality of the separation.

How Gas Chromatography work?

Like for all other chromatographic techniques, a mobile and a stationary phase are required.

• The mobile phase (carrier gas) is comprised of an inert gas e.g. helium, argon, nitrogen, etc.
• The stationary phase consists of a packed /capillary column where the packing or solid support itself acts as stationary phase, or is coated with the liquid stationary phase (high boiling polymer).
• More commonly used in many applications are capillary columns, where the stationary phase coats the walls of a small-diameter tube directly (e.g. 0.25 mm film in a 0.32 mm tube).
• The stronger the interaction is the longer the compound remains attached to the stationary phase, and the more time it takes to go through the column (longer retention time).

10 Tips to Improve Your Speaking Voice

One of the most important components of public speaking is the sound of your voice. It influences the impact of your message, and might even make or break the success of your speech. Fortunately, for many people, good voice quality can be learned.
Instructions :
Breathe from your diaphragm - Practice long and controlled exhales. When you speak, use breath to punctuate your point. For example, take a breath at the end of each phrase whether you need to or not. Use that opportunity to pause and let the listeners absorb what you say.
Use pitch - Lower pitches generally are more soothing to hear. However, modulating your pitch for emphasis will keep your listeners engaged. Develop your pitch by practicing humming.
Moderate your volume - Find out if you speak too loudly or too softly. When you begin speaking, ask your audience how your volume is (each situation is different). Try to stay at the appropriate volume throughout your speech.
Moderate your pace - This one is also closely related to breath. If you speak too quickly, people can’t keep up. If you speak too slowly, people will lose interest. Record your speech to determine if you need to change your pace. Get feedback from others.
Articulate - Try exaggerating your lip movement to reduce mumbling. Practice articulating tongue twisters and extending and exaggerating vowel sounds. Become an expert at articulating tongue twisters as quickly and crisply as possible. Focus on the ones you find difficult.
Practice - your speech in advance and determine where you want to pause for a breath. For more emphasis, pause for more than one breath. Mark your breathing points in your notes.
Loosen up - before you begin. Look side to side. Roll your head in half-circles and roll your shoulders back. Shift your rib cage from side to side. Yawn. Stretch. Touch your toes while completely relaxing your upper body, then slowly stand up, one vertebra at a time, raising your head last. Repeat as needed.
Posture - Stand up straight and tall to allow full lung capacity and airflow.
Record - your voice repeatedly using different ways of speaking. Determine which one is most pleasing.
Practice breath control - Take a deep breath, and while you exhale, count to 10 (or recite the months or days of the week). Try gradually increasing your volume as you count, using your abdominal muscles—not your throat—for volume. Don’t let your larynx tense up.

Accreditation and Certification

The terms "accreditation" and "certification" are sometimes used interchangeably, however, they are not synonymous.

Certification is used for verifying that personnel have adequate credentials to practice certain disciplines, as well as for verifying that products meet certain requirements.

Accreditation is used to verify that laboratories have an appropriate quality management system and can properly perform certain test methods (e.g., ANSI, ASTM, and ISO test methods) and calibration parameters according to their scopes of accreditation.