Application of Machine

7/30/2019 Application of Machine

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Kod Subjek : GTK 304

Nama : Noor Izwan bin Noor Zaimi

No. Matrik : 109511

Program : KPP Tahun 3


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1.High-performance liquid chromatography

History of HPLC

Liquid chromatography was initially discovered as an analytical technique in the early

twentieth century and was first used as a method of separating colored compounds. This is

where the name chromatography chroma means color, graphy means writing, was derived.

A Russian botanist named Mikhail S. Tswett used a rudimentary form of chromatographic

separation to purify mixtures of plant pigments into the pure constituents. He separated the

pigments based on their interaction with a stationary phase, which is essential to any

chromatographic separation. The stationary phase he used was powdered chalk and

aluminia, the mobile phase in his separation was the solvent. After the solid stationary phase

was packed into a glass column (essentially a long, hollow, glass tube) he poured the

mixture of plant pigments and solvent in the top of the column. He then poured additional

solvent into the column until the samples were eluted at the bottom of the column. The result

of this process most crucial to his investigation was that the plant pigments separated into

bands of pure components as they passed through the stationary phase. Modern high

performance liquid chromatography or HPLC has its roots in this separation, the first form

of liquid chromatography. The chromatographic process has been significantly improved

over the last hundred years, yielding greater separation efficiency, versatility and speed.

Chromatographic Theory

Affinities for Mobile and Stationary Phases

All chromatographic separations, including HPLC operate under the same basic principle;

every compound interacts with other chemical species in a characteristic

manner. Chromatography separates a sample into its constituent parts because of the

difference in the relative affinities of different molecules for the mobile phase and the

stationary phase used in the separation.

Distribution Constant

All chemical reactions have a characteristic equilibrium constant. There is a chemical

equilibrium constant Keq that dictates what percentage of compound A will be in solution and

what percentage will be bound to the stationary compound B. During a chromatographic

separation, there is similar relationship between compound A and the solvent, or mobile

phase, C. This will yield an overall equilibrium equation which dictates the quantity of A that

will be associated with the stationary phase and the quantity of A that will be associated with

the mobile phase.

The equilibrium between the mobile phase and stationary phase is given by the constant K c.


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Where Kc, the distribution constant, is the ratio of the activity of compound A in the stationary

phase and activity of compound A in the mobile phase. In most separations, which contain

low concentrations of the species to be separated, the activity of A in each is approximately

equal to the concentration of A in that state. The distribution constant indicates the amount

of time that compound Aspends adsorbed to the stationary phase as the opposed to the

amount of time A spends solvated by the mobile phase. This relationship determines the

amount of time it will take for compound A to travel the length of the column. The more

time A spends adsorbed to the stationary phase, the more time compound A will take to

travel the length of the column. The amount of time between the injection of a sample and its

elution from the column is known as the retention time; it is given the symbol tR.

The amount of time required for a sample that does not interact with the stationary phase, or

has a Kc equal to zero, to travel the length of the column is known as the void time, t M. No

compound can be eluted in less than the void time.

Retention Factor

Since Kc is a factor that is wholly dependent on a particular column and solvent flow rate, a

quantitative measure of the affinity of a compound for a particular set of mobile and

stationary phases that does not depend on the column geometry is useful. The retention

factor, k, can be derived from Kc and is independent of the column size and the solvent flow

rate.


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The retention factor is calculated by multiplying the distribution constant by the volume of

stationary phase in the column and dividing by the volume of mobile phase in the column.

Selectivity

In order to separate two compounds, their respective retention factors must be different,

otherwise both compounds would be eluted simultaneously; the selectivity factor is the ratio

of the retention factors.

Where B is the compound that is retained more strongly by the column and A is the

compound with the faster elution time.

Band Broadening

As a compound passes through the column it slowly diffuses away from the initial injection

band, which is the area of greatest concentration. The initial, narrow, band that contained all

of the sample becomes broader the longer the analyte remains in the column. This band

broadening increases the time required for complete elution of a particular compound and is

generally undesirable. It must be minimized so that overly broad elution bands do not

overlap with one another. We will see how this is measured quantitatively when we

discuss peak resolution momentarily.

Separation Efficiency

The overriding purpose of a chromatographic separation is just that, to separate two or more

compounds contained in solution. In analytical chemistry, a quantitative metric of every

experimental parameter is desired, and so separation efficiency is measured in plates. The

concept of plates as a separation metric arose from the original method of fractional

distillation, where compounds were separated based on their volatilities through many

simultaneous simple distillations, each simple distillation occurred on one of many distillation

plates. In chromatography, no actual plates are used, but the concept of a theoretical plate,

as a distinct region where a single equilibrium is maintained, remains. In a

particular liquid chromatographic separation, the number of theoretical plates and theheight

equivalent to a theoretical plate are related simply by the length of the column.

Where N is the number of theoretical plates, L is the length of the column, and H is the

height equivalent to a theoretical plate. The plate height is given by the variance (standard

deviation squared) of an elution peak divided by the length of the column.

The standard deviation of an elution peak can be approximated by assuming that a

Gaussian elution peak is roughly triangular, in that case the plate height can be given by the

width of the elution peak squared times the length of the column over the retention time of

the that peak squared times 16.


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Using the relationship between plate height and number of plates, the number of plates can

also be found in terms of retention time and peak width.

In order to optimize separation efficiency, it is necessary in maximize the number of

theoretical plates, which requires reducing the plate height. The plate height is related to

the flow rate of the mobile phase, so for a fixed set of mobile phase, stationary phase, and

analytes; separation efficiency can be maximized by optimizing flow rate as dictated by

the van Deemter equation.

The three constants in the van Deemter equation are factors that describe possible causes

of band broadening in a particular separation. A is a constant which represents the different

possible paths that can be taken by the analyte through the stationary phase, it decreases if

the packing of the column is kept as small as possible. B is a constant that describes the

longitudinal diffusion that occurs in the system. C is a constant that describes the rate of

adsorption and desorption of the analyte to the stationary phase. A, Band C are constant for

any given system (with constant analyte, stationary phase, and mobile phase), so flow rate

must be optimized accordingly. If the flow rate is too low, the longitudinal diffusion

factor (B/v) will increase significantly, which will increase plate height. At low flow rates, the

analyte spends more time at rest in the column and therefore longitudinal diffusion in a more

significant problem. If the flow rate is too high, the mass transfer term (C*v) will increase and

reduce column efficiency. At high flow rates the adsorption of the analyte to the stationary

phase results in some of the sample lagging behind, which also leads to band broadening.

Resolution

The resolution of a elution is a quantitative measure of how well two elution peaks can be

differentiated in a chromatographic separation. It is defined as the difference in retention

times between the two peaks, divided by the combined widths of the elution peaks.

Where B is the species with the longer retention time, and tR and W are the retention time

and elution peak width respectively. If the resolution is greater than one, the peaks can

usually be differentiated successfully.


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HPLC as a solution to efficiency problems

While all of these basic principles hold true for all chromatographic separations, HPLC was

developed as method to solve some of the shortcomings of standard liquid chromatography.

Classic liquid chromatography has several severe limitations as a separation method. When

the solvent is driven by gravity, the separation is very slow, and if the solvent is driven by

vacuum, in a standard packed column, the plate height increases and the effect of the
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vacuum is negated. The limiting factor in liquid chromatography was originally the size of the

column packing, once columns could be packed with particles as small as 3 m, faster

separations could be performed in smaller, narrower, columns. High pressure was required

to force the mobile phase and sample through these new columns, and previously unneeded

apparatus was required to maintain reproducibility of results in this new instruments. The use

of high pressures in a narrow column allowed for a more effective separation to be achieved

in much less time than was required for previous forms of liquid chromatography.

Apparatus

Specialized apparatus is required for an HPLC separation because of the high pressures

and low tolerances under which the separation occurs. If the results are to be reproducible,

then the conditions of the separation must also be reproducible. Thus HPLC equipment must

be of high quality; it is therefore expensive.

Solvent

The mobile phase, or solvent, in HPLC is usually a mixture of polar and non-

polar liquid components whose respective concentrations are varied depending on the

composition of the sample. As the solvent is passed through a very narrow bore column, any

contaminants could at worst plug the column, or at the very least add variability to the

retention times during repeated different trials. Therefore HPLC solvent must be kept free of

dissolved gases, which could come out of solution mid-separation, and particulates.

Column

In the HPLC column, the components of the sample separate based on their differing

interactions with the column packing. If a species interacts more strongly with the stationary

phase in the column, it will spend more time adsorbed to the column's adsorbent and will

therefore have a greater retention time. Columns can be packed with solids such as silica or

alumina; these columns are calledhomogeneous columns. If stationary phase in the column

is a liquid, the column is deemed a bonded column. Bonded columns contain

a liquid stationary phase bonded to a sold support, which is again usually silica or alumina.

The value of the constant Cdescribed in the van Deemter equation is proportional, in HPLC,

to the diameter of the particles that constitute the column's packing material.

Pump

The HPLC pump drives the solvent and sample through the column. To reduce variation in

the elution, the pump must maintain a constant, pulse free, flow rate; this is achieved

with multi-piston pumps. The presence of two pistons allows the flow rate to be controlled byone piston as the other recharges. A syringe pump can be used for even greater control of


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time. A sample containing compounds of a wide range of polarities can be separated by a

gradient elution in a shorter time period without a loss of resolution in the earlier peaks or

excessive broadening of later peaks. However, gradient elution requires more complex and

expensive equipment and it is more difficult to maintain a constant flow rate while there are

constant changes in mobile phase composition. Gradient elution, especially at high speeds,

brings out the limitations of lower quality experimental apparatus, making the results

obtained less reproducible in equipment already prone to variation. If the flow rate or mobile

phase composition fluctuates, the results will not be reproducible.

Applications

HPLC can be used in both qualitative and quantitative applications, that is for both

compound identification and quantification. Normal phase HPLC is only rarely used now,

almost all HPLC separation can be performed in reverse phase. Reverse phase HPLC

(RPLC) is ineffective in for only a few separation types; it cannot separate inorganic ions

(they can be separated by ion exchange chromatography). It cannot separate

polysaccharides (they are too hydrophilic for any solid phase adsorption to occur), nor

polynucleotides (they adsorb irreversibly to the reverse phase packing). Lastly, incredibly

hydrophobic compounds cannot be separated effectively by RPLC (there is little selectivity).

Aside from these few exceptions, RPLC is used for the separation of almost all other

compound varieties. RPLC can be used to effectively separate similar simple and aromatic

hydrocarbons, even those that differ only by a single methylene group. RPLC effectively

separates simple amines, sugars, lipids, and even pharmaceutically active compounds.

RPLC is also used in the separation of amino acids, peptides, and proteins. Finally RPLC is

used to separate molecules of biological origin. The determination of caffeine content in

coffee products is routinely done by RPLC in commercial applications in order to guarantee

purity and quality of ground coffee. HPLC is a useful addition to an analytical arsenal,

especially for the separation of a sample before further analysis.


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2.Atomic absorption spectroscopy

Atomic absorption absorption spectroscopy (AA or AAS) is one of the commonest

instrumental methods for analyzing for metals and some metalloids.

Application :

water analysis (e.g. Ca, Mg, Fe, Si, Al, Ba content)

food analysis

analysis of animal feedstuffs (e.g. Mn, Fe, Cu, Cr, Se, Zn)

analysis of additives in lubricating oils and greases (Ba, Ca, Na, Li, Zn, Mg)

analysis of soils

clinical analysis (blood samples: whole blood, plasma, serum; Ca, Mg, Li, Na, K, Fe)

The Hollow Cathode Lamp

The hollow cathode lamp (HCL) uses a cathode made of the element of interest with a low

internal pressure of an inert gas. A low electrical current (~ 10 mA) is imposed in such a way

that the metal is excited and emits a few spectral lines characteristic of that element (for

instance, Cu 324.7 nm and a couple of other lines; Se 196 nm and other lines, etc.). The

light is emitted directionally through the lamp's window, a window made of a glass

transparent in the UV and visible wavelengths.

Neublizer, Different Oxidants, and Burner Heads, and Waste

The nebulizer chamber thoroughly mixes acetylene (the fuel) and oxidant (air or nitrous

oxide), and by doing so, creates a negative pressure at the end of the small diameter, plastic


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nebulizer tube (not shown in adjacent figure; see figure below). This negative pressure acts

to suck ("uptake") liquid sample up the tube and into the nebulizer chamber, a process called

aspiration. A small glass impact bead and/or a fixed impeller inside the chamber creates a

heterogeneous mixture of gases (fuel + oxidant) and suspended aerosol (finely dispersed

sample). This mixture flows immediately into the burner head where it burns as a smooth,

laminar flame evenly distributed along a narrow slot in the well-machined metal burner head.

Liquid sample not flowing into the flame collects on the bottom of the nebulizer chamber and

flows by gravity through a waste tube to a glass waste container (remember, this is still

highly acidic).

For some elements that form refractory oxides (molecules hard to break down in the flame)

nitrous oxide (N2O) needs to be used instead of air (78% N2 + 21% O2) for the oxidant. In

that case, a slightly different burner head with a shorter burner slot length is used.

The Monochromator and PMT

Tuned to a specific wavelength and with a specified slit width chosen, the monochromator

isolates the hollow cathode lamp's analytical line. Since the basis for the AAS process is

atomic ABSORPTION, the monochromator seeks to only allow the light not absorbed by the

analyte atoms in the flame to reach the PMT. That is, before an analyte is aspirated, a

measured signal is generated by the PMT as light from the HCL passes through the flame.

When analyte atoms are present in the flame--while the sample is aspirated--some of that

light is absorbed by those atoms (remember it is not the ionic but elemental form that

absorbs). This causes a decrease in PMT signal that is proportional to the amount of

analyte. This last is true inside the linear range for that element using that slit and that

analytical line. The signal is therefore a decrease in measure light:

atomic absorption spectroscopy.


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Acidic Content and Oxidation State of Samples and Standards

The samples and standards are often prepared with duplicate acid concentrations to

replicate the analyte's chemical matrix as closely as possible. Acid contents of 1% to 10%

are common.

In addition, high acid concentrations help keep all dissolved ions in solution.

The oxidation state of the analyte metal or metalloid is important in AAS. For instance, AAS

analysis of selenium requires the Se(IV) oxidation state (selenite). Se(VI), the more highly

oxidized state of the element (selenate), responds erratically and non reproducibly in the

system. Therefore, all selenium in Se calibration standards and Se containing samples must

be in the Se(IV) form for analysis. This can be accomplished by oxidizing all Se in the

sample to selenate using a strong oxidizer such as nitric acid or hydrogen peroxide and then

reducing the contained selenate to selenite with boiling HCl.

Double Beam Instruments

The light from the HCL is split into two paths using a rotating mirror: one pathway passes

through the flame and another around. Both beams are recombined in space so they both hit

the PMT but separated in time. The beams alternate quickly back and forth along the two

paths: one instant the PMT beam is split by the rotating mirror and the sample beam passes

through the flame and hits the PMT. The next instance, the HCL beam passes through a

hole in the mirror and passes directly to the PMT without passing through the flame. The

difference between these beams is the amount of light absorbed by atoms in the flame.

The purpose of a double beam instrument is to help compensate for drift of the output of the

hollow cathode lamp or PMT. If the HCL output drifts slowly the subtraction process

described immediately above will correct for this because both beams will drift equally on the

time scale of the analysis. Likewise if the PMT response changes the double beam

arrangement take this into account.

Ignition, Flame conditions, and Shut Down

The process of lighting the AAS flame involves turning on first the fuel then the oxidant and

then lighting the flame with the instrument's auto ignition system (a small flame or red-hot

glow plug). After only a few minutes the flame is stable. Deionized water or a dilute acid


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solution can be aspirated between samples. An aqueous solution with the correct amount of

acid and no analyte is often used as the blank.

Careful control of the fuel/air mixture is important because each element's response

depends on that mix in the burning flame. Remember that the flame must breakdown the

analyte's matrix and reproducibly create the elemental form of the analyte atom.

Optimization is accomplished by aspirating a solution containing the element (with analyte

content about that of the middle of the linear response range) and then adjusting the

fuel/oxidant mix until the maximum light absorbance is achieved. Also the position of the

burned head and nebulizer uptake rate are similarly "tuned." Most computer controlled

systems can save variable settings so that methods for different elements can be easily

saved and reloaded.

Shut down involves aspirating deionized water for a short period and then closing the fuel off

first. Most modern instruments control the ignition and shutdown procedures automatically.

3.Scanning electron microscope

A scanning electron microscope (SEM) is a type ofelectron microscope that produces

images of a sample by scanning it with a focused beam ofelectrons. The electrons interactwith electrons in the sample, producing various signals that can be detected and that contain

information about the sample's surface topography and composition. The electron beam is

generally scanned in a raster scan pattern, and the beam's position is combined with the

detected signal to produce an image. SEM can achieve resolution better than 1 nanometer.

Specimens can be observed in high vacuum, low vacuum and in environmental SEM

specimens can be observed in wet conditions.

The types of signals produced by a SEM include secondary electrons (SE), back-scattered

electrons (BSE), characteristic X-rays, light (cathodoluminescence) (CL), specimen current

and transmitted electrons. Secondary electron detectors are standard equipment in all

SEMs, but it is rare that a single machine would have detectors for all possible signals. The

signals result from interactions of the electron beam with atoms at or near the surface of the

sample. In the most common or standard detection mode, secondary electron imaging or

SEI, the SEM can produce very high-resolution images of a sample surface, revealing

details less than 1 nm in size. Due to the very narrow electron beam, SEM micrographs have

a largedepth of field yielding a characteristic three-dimensional appearance useful forunderstanding the surface structure of a sample. This is exemplified by the micrograph of
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pollen shown above. A wide range of magnifications is possible, from about 10 times (about

equivalent to that of a powerful hand-lens) to more than 500,000 times, about 250 times the

magnification limit of the best light microscopes.

Back-scattered electrons (BSE) are beam electrons that are reflected from the sample

by elastic scattering. BSE are often used in analytical SEM along with the spectra made from

the characteristic X-rays, because the intensity of the BSE signal is strongly related to the

atomic number (Z) of the specimen. BSE images can provide information about the

distribution of different elements in the sample. For the same reason, BSE imaging can

imagecolloidal gold immuno-labels of 5 or 10 nm diameter, which would otherwise be difficult

or impossible to detect in secondary electron images in biological specimens.

Characteristic X-rays are emitted when the electron beam removes an inner shell

electron from the sample, causing a higher-energy electron to fill the shell and releaseenergy. These characteristic X-rays are used to identify the composition and measure the

abundance of elements in the sample.

All samples must also be of an appropriate size to fit in the specimen chamber and are

generally mounted rigidly on a specimen holder called a specimen stub. Several models of

SEM can examine any part of a 6-inch (15 cm) semiconductor wafer, and some can tilt an

object of that size to 45.

For conventional imaging in the SEM, specimens must be electrically conductive, at least at

the surface, and electrically grounded to prevent the accumulation ofelectrostatic charge at

the surface. Metal objects require little special preparation for SEM except for cleaning and

mounting on a specimen stub. Nonconductive specimens tend to charge when scanned by

the electron beam, and especially in secondary electron imaging mode, this causes

scanning faults and other image artifacts. They are therefore usually coated with an ultrathin

coating of electrically conducting material, deposited on the sample either by low-

vacuum sputter coating or by high-vacuum evaporation. Conductive materials in current use

for specimen coating include gold,gold/palladium alloy, platinum, osmium, iridium, tungsten, chromium, and graphite.

Additionally, coating may increase signal/noise ratio for samples of low atomic number(Z).

The improvement arises because secondary electron emission for high-Z materials is

enhanced.

An alternative to coating for some biological samples is to increase the bulk conductivity of

the material by impregnation with osmium using variants of the OTO staining method (O-

osmium, T-thiocarbohydrazide, O-osmium).
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Nonconducting specimens may be imaged uncoated using environmental SEM (ESEM) or

low-voltage mode of SEM operation. Environmental SEM instruments place the specimen in

a relatively high-pressure chamber where the working distance is short and the electron

optical column is differentially pumped to keep vacuum adequately low at the electron gun.

The high-pressure region around the sample in the ESEM neutralizes charge and provides

an amplification of the secondary electron signal. Low-voltage SEM is typically conducted in

an FEG-SEM because the field emission guns (FEG) is capable of producing high primary

electron brightness and small spot size even at low accelerating potentials. Operating

conditions to prevent charging of non-conductive specimens must be adjusted such that the

incoming beam current was equal to sum of outcoming secondary and backscattered

electrons currents. It usually occurs at accelerating voltages of 0.34 kV.

Embedding in a resin with further polishing to a mirror-like finish can be used for bothbiological and materials specimens when imaging in backscattered electrons or when doing

quantitative X-ray microanalysis.

The main preparation techniques are not required in the environmental SEM outlined below,

but some biological specimens can benefit from fixation.

In a typical SEM, an electron beam is thermionically emitted from an electron gun fitted with

a tungsten filament cathode. Tungsten is normally used in thermionic electron guns because

it has the highest melting point and lowest vapour pressure of all metals, thereby allowing it

to be heated for electron emission, and because of its low cost. Other types of electron

emitters include lanthanum hexaboride (LaB6) cathodes, which can be used in a standard

tungsten filament SEM if the vacuum system is upgraded and FEG, which may be of

the cold-cathode type using tungsten single crystal emitters or the thermally

assistedSchottky type, using emitters ofzirconium oxide.

The electron beam, which typically has an energy ranging from 0.2 keV to 40 keV, is focused

by one or two condenser lenses to a spot about 0.4 nm to 5 nm in diameter. The beam

passes through pairs of scanning coils or pairs of deflector plates in the electron column,

typically in the final lens, which deflect the beam in the xand yaxes so that it scans in

a rasterfashion over a rectangular area of the sample surface.

When the primary electron beam interacts with the sample, the electrons lose energy by

repeated random scattering and absorption within a teardrop-shaped volume of the

specimen known as the interaction volume, which extends from less than 100 nm to around

5 m into the surface. The size of the interaction volume depends on the electron's landing

energy, the atomic number of the specimen and the specimen's density. The energy

exchange between the electron beam and the sample results in the reflection of high-energy
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electrons by elastic scattering, emission of secondary electrons by inelastic scattering and

the emission ofelectromagnetic radiation, each of which can be detected by specialized

detectors. The beam current absorbed by the specimen can also be detected and used to

create images of the distribution of specimen current. Electronic amplifiers of various types

are used to amplify the signals, which are displayed as variations in brightness on a

computer monitor (or, for vintage models, on a cathode ray tube). Each pixel of computer

videomemory is synchronized with the position of the beam on the specimen in the

microscope, and the resulting image is therefore a distribution map of the intensity of the

signal being emitted from the scanned area of the specimen. In older microscopes image

may be captured by photography from a high-resolution cathode ray tube, but in modern

machines image is saved to a computer data storage.

4.X-ray photoelectron spectroscopy

X-ray photoelectron spectroscopy (XPS) is a quantitative spectroscopic technique that

measures the elemental composition, empirical formula, chemical state and electronic

state of elements that exist within a material. XPS spectra are obtained by irradiating a

material with a beam of X-rays while simultaneously measuring the kinetic energy and

number of electrons that escape from the top 1 to 10 nm of the material being analyzed.

XPS requires ultra-high vacuum (UHV) conditions.

XPS is a surface chemical analysis technique that can be used to analyze the surface

chemistry of a material in its "as received" state, or after some treatment, for example:

fracturing, cutting or scraping in air or UHV to expose the bulk chemistry, ion beam

etching to clean off some of the surface contamination, exposure to heat to study the

changes due to heating, exposure to reactive gases or solutions, exposure to ion beam

implant, exposure to ultraviolet light.
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XPS is used to measure:

elemental composition of the surface (top 110 nm usually)

empirical formula of pure materials

elements that contaminate a surface

chemical or electronic state of each element in the surface

uniformity of elemental composition across the top surface (or line profiling or

mapping)

uniformity of elemental composition as a function of ion beam etching (or depth

profiling)

Figure 5.0: Basic components of XPS

A surface is irradiated with X-rays (commonly Al K or Mg K) in vacuum. When an x-ray

photon hits and transfers this energy to a core-level electron, it is emitted from its initial state

with a kinetic energy dependent on the incident X-ray and binding energy of the atomic

orbital from which it originated. The energy and intensity of the emitted photoelectrons are

analysed to identify and determine the concentrations of the elements present. Thesephotoelectrons originate from a depth of


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5.Gas chromatography

Some of the different applications of gas chromatography where it can be used such as:

Used in pharmaceuticals

used in pollutants petroleum

petrochemicals

oils

fats

food and flavors

vitamins

steroids and alkaloids

blood and serum

pesticides and fungicides

radioactive isotopes

used in cosmetics

used in environmental toxins.

A gas chromatograph is a chemical analysis instrument for separating chemicals in a

complex sample. A gas chromatograph uses a flow-through narrow tube known asthe column, through which different chemical constituents of a sample pass in a gas stream

(carrier gas, mobile phase) at different rates depending on their various chemical and

physical properties and their interaction with a specific column filling, called the stationary

phase. As the chemicals exit the end of the column, they are detected and identified

electronically. The function of the stationary phase in the column is to separate different

components, causing each one to exit the column at a different time (retention time). Other

parameters that can be used to alter the order or time of retention are the carrier gas flow

rate, column length and the temperature.

In a GC analysis, a known volume of gaseous or liquid analyte is injected into the "entrance"

(head) of the column, usually using a microsyringe (or, solid phase microextraction fibers, or

a gas source switching system). As the carrier gas sweeps the analyte molecules through

the column, this motion is inhibited by the adsorption of the analyte molecules either onto the

column walls or onto packing materials in the column. The rate at which the molecules

progress along the column depends on the strength ofadsorption, which in turn depends on

the type of molecule and on the stationary phase materials. Since each type of molecule has

a different rate of progression, the various components of the analyte mixture are separated
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as they progress along the column and reach the end of the column at different times

(retention time). A detector is used to monitor the outlet stream from the column; thus, the

time at which each component reaches the outlet and the amount of that component can be

determined. Generally, substances are identified (qualitatively) by the order in which they

emerge (elute) from the column and by the retention time of the analyte in the column.

The method is the collection of conditions in which the GC operates for a given

analysis. Method development is the process of determining what conditions are adequate

and/or ideal for the analysis required.

Conditions which can be varied to accommodate a required analysis include inlet

temperature, detector temperature, column temperature and temperature program, carrier

gas and carrier gas flow rates, the column's stationary phase, diameter and length, inlet type

and flow rates, sample size and injection technique. Depending on the detector installed on

the GC, there may be a number of detector conditions that can also be varied. Some GCs

also include valves which can change the route of sample and carrier flow. The timing of the

opening and closing of these valves can be important to method development.


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6.X-Ray Diffractometer

Applications of XRD

Pharmaceutical industry

X-ray diffraction (XRD) can be used to unambiguously characterize the composition of

pharmaceuticals. An XRD-pattern is a direct result of the crystal structures, which are

present in the pharmaceutical under study. As such, the parameters typically associated with

crystal structure can be simply accessed. For example, once an active drug has been

isolated, an indexed X-ray powderdiffraction pattern is required to analyse the crystal

structure, secure a patent and protect the companys investment.

For multi-component formulations, the actual percentages of the active ingredients in the

final dosage form can be accurately analysed in situ, along with the percentage of

any amorphous packing ingredients used.

XRD is the key technique for solid-state drug analysis, benefiting all stages of drug

development, testing and production.

Forensic science

XRD is used mainly in contact trace analysis. Examples of contact traces are paint flakes,

hair, glass fragments, stains of any description and loose powdered materials. Identification

and comparison of trace quantities of material can help in the conviction or exoneration of a

person suspected of involvement in a crime.

Geological applications

XRD is the key tool in mineral exploration. Mineralogists have been amongst the foremost to

develop and promote the new field of X-ray crystallography after its discovery. Thus, the

advent of XRD has literally revolutionized the geological sciences to such a degree that they

have become unthinkable without this tool. Nowadays, any geological group actively

involved in mineralogical studies would be lost without XRD to unambiguously characterise

the individual crystal structures. Each mineral type is defined by a characteristic crystal

structure, which will give a unique x-ray diffraction pattern, allowing rapid identification of

minerals present within a rock or soil sample. The XRD data can be analysed to determine

the proportion of the different minerals present.
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Microelectronics industry

As the microelectronics industry uses silicon and gallium arsenide single crystal substrates

in integrated circuit production, there is a need to fully characterise these materials using the

XRD. XRD topography can easily detect and image the presence of defects within a crystal,making it a powerful non-destructive evaluation tool for characterising industrially important

single crystal specimens.

Glass industry

While glasses are X-ray amorphous and do not themselves give X-ray diffraction patterns,

there are still manifold uses of XRD in the glass industry. They include identification

ofcrystalline particles which cause tiny faults in bulk glass, and measurements of crystalline

coatings for texture, crystallite size and crystallinity.
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Application of Machine

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