Analytical Chemistry Measuring The Chemical Composition Biology Essay

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Analytical chemistry is the science that seeks ever improved means of measuring the chemical composition of natural and artificial materials. Chemical composition is the entire picture (composition) of the material at the chemical scale and includes geometric features such as molecular morphologies and distributions of species within a sample as well as single dimensional features such as percent composition and species identity.

To be effective and efficient, analyzing samples requires expertise in

The chemistry that can occur in a sample.

Analysis and sample handling methods for a wide variety of problems (the tools-of-the-trade).

Accuracy and precision of the method.

Proper data analysis and record keeping.

The major stages of an analytical process are described as follows:

Sample preparation

Sample analysis

Data handling

Sample transport and storage

Archiving

Report generation

Information to customer

Sample collection and sampling

Steps in analytical cycle

The pharmaceutical analysis comprises the procedures necessary to determine the "identity, strength, quality and purity" of such compounds. It also includes the analysis of raw material and intermediates during manufacturing process of drugs.

Types

Qualitative analysis

 Qualitative inorganic analysis seeks to establish the presence of a given element or inorganic compound in a sample.

 Qualitative organic analysis seeks to establish the presence of a given functional group or organic compound in a sample.

Quantitative analysis

 Quantitative analysis seeks to establish the amount of a given element or compound in a sample.

Methods of detecting analytes

physical means

 Mass

 Color

 Refractive index

 Thermal conductivity

with electromagnetic radiation (Spectroscopy)

 Absorption

 Emission

 Scattering

by an electric charge

 Electrochemistry

 Mass spectrometry

Traditional analytical techniques

Titration

Titration involves the addition of a reactant to a solution being analyzed until some equivalence point is reached. Often the amount of material in the solution being analyzed may be determined.

Gravimetry

Gravimetric analysis involves determining the amount of material present by weighing the sample before and/or after some transformation. A common example used in the determination of the amount of water in a hydrate by heating the sample to remove the water such that the difference in weight is due to the water lost.

Instrumental Analysis

Block diagram of an analytical instrument showing the stimulus and measurement of response

1. Spectroscopy

Spectroscopy measures the interaction of the molecules with electromagnetic radiation. Spectroscopy consists of many different applications such as

 Atomic absorption spectroscopy

 Atomic emission spectroscopy

 Ultraviolet-visible spectroscopy

 Infrared spectroscopy

 Raman spectroscopy

 Nuclear magnetic resonance spectroscopy

 Photoemission spectroscopy

 Mössbauer spectroscopy, etc.

2. Mass Spectrometry

Mass spectrometry measures mass-to-charge ratio of molecules using electric and magnetic fields. There are several ionization methods: electron impact, chemical ionization, electrospray, matrix assisted laser desorption ionization, and others. Also, mass spectrometry is categorized by approaches of mass analyzers: magnetic-sector,quadrupole mass analyzer, quadrupole ion trap, time-of-flight, fourier transform ion cyclotron resonance, etc.

3. Crystallography

Crystallography is a technique that characterizes the chemical structure of materials at the atomic level by analyzing the diffraction patterns of usually x-rays that have been deflected by atoms in the material. From the raw data the relative placement of atoms in space may be determined.

4. Electrochemical Analysis

Electrochemistry measures the interaction of the material with an electric field.

5. Thermal Analysis

Calorimetry and thermogravimetric analysis measure the interaction of a material and heat.

6. Separation

Separation processes are used to decrease the complexity of material mixtures. Chromatography and electrophoresis are representative of this field.

Chromatographic methods

Chromatography is a technique by which the components in a sample, carried by the liquid or gaseous phase, are resolved by sorption-desorption steps on the stationary phase.

There are various advanced chromatographic techniques, which are most reliable and widely used for the estimation of multicomponent drugs in their formulation namely,

Gas chromatography (GC)

High Performance Thin Layer Chromatography (HPTLC)

High Performance Liquid Chromatography (HPLC)

High performance liquid chromatographic separation is based on interaction and differential partition of the sample between the mobile phase and stationary phase. The commonly used chromatographic methods can be roughly divided into the following groups,

Chiral

Ion-exchange

Ion pair/affinity

Normal phase

Reverse phase

Size exclusion

When compared to classical column chromatography, this technique is preferred because of its improved performance in terms of rapidity, specificity, sensitivity, accuracy, covenience, ease of automation and the cost of analysis.

Advance in column technology, high pressure pumping system and sensitive detectors have transformed liquid column chromatography into a high speed, efficient, accurate and highly resolved method of separation.

7. Hybrid Techniques

Combination of the above techniques produces 'hybrid' or 'hyphenated' techniques. Several examples are in popular use today and new hybrid techniques are under development. For example

GC-MS

LC-MS

HPLC/ ESI-MS

LC-DAD

CE-MS

CE-UV

8. Microscopy

The visualization of single molecules, single cells, biological tissues and nano- micro materials is very important and attractive approach in analytical science. Also, hybridization with other traditional analytical tools is revolutionizing analytical science. Microscopy can be categorized into three different fields: optical microscopy, electron microscopy, and scanning probe microscopy. Recently, this field is rapidly progressing because of the rapid development of computer and camera industries.

9. Lab-on-a-chip

Miniaturized analytical instrumentation, which is also called as microfluidics or micro total analysis system (μTAS). The beauty of lab-on-a-chip system is that a whole device can be visualized under a microscope.

Method of data analysis

a) Standard Curve

A standard method for analysis of concentration involves the creation of a calibration curve. This allows for determination of the amount of a chemical in a material by comparing the results of unknown sample to those of a series known standards.

b) Internal Standard

Sometimes an internal standard is added at a known concentration directly to an analytical sample to aid in quantitation. The amount of analyte present is then determined relative to the internal standard as a calibrant.

Quality assurance plays a central role in determining the safety and efficacy of medicines. Highly specific and sensitive analytical technique holds the key to design, development, standardization and quality control of medicinal products.

The efficacy and safety of a medicinal product can be assured by analytical monitoring of its quality. It is important that analytical procedure proposed of a particular active ingredient or its dosage form should be systematically sound under the condition in which it is to be applied.

New Drug Discovery

New drugs have been discovered from two major sources

 Synthetic chemicals

 Natural products including plants, animal and microbes.

The number of drugs introduced into the market has been increasing at an alarming rate. Newer analytical methods are developed for these drugs or drug combinations because of the following reasons

The drug or combination may not be official in any pharmacopoeia.

A literature search may not reveal an analytical procedure for the drug or its combination.

Analytical methods may not be available for the drug combination due to the interference caused by excipients.

Analytical methods for the quantification of drug or drug combination with other drugs may not be available.

On the other hand, the existing procedure may

 Require expensive instruments, reagents, solvents etc.

 Involve any tedious extraction or separation steps which may be quite time consuming.

 Not be rapid, reliable or sensitive.

The newly developed analytical methods find their importance in various fields such as

 Research institutions

 Quality control department in industries

 Approved testing laboratories

 Bio-pharmaceutical and bio-equivalence studies

 Clinical pharmacokinetic studies

INTRODUCTION TO UV ABSORPTION SPECTROPHOTOMETRY

Ultraviolet-visible spectrophotometry is one of the most frequently employed techniques in pharmaceutical analysis. It involves the measurement of the amount of ultraviolet radiations in the range of

200-400 nm or 2000-4000 Ǻ absorbed by a substance in solution. The UV radiation has sufficient energy to excite valence electrons in many atoms or molecules. Consequently UV is involved with electronic excitation. Sometimes because of this electronic excitation, ultraviolet spectroscopy is also known as electronic spectroscopy. The measurement of absorption of ultraviolet radiation provides a convenient means for the analysis of numerous inorganic and organic species.

Absorption of light in ultraviolet region of the electromagnetic spectrum occurs when the energy of light matches that required to induce in the molecule an electronic transition and its associated vibrational and rotational transitions.

A compound or drug which possesses conjugated double bond absorbs UV radiation at a specific wavelength and this character of drug is specific for a fixed solvent system.

The wavelength at which maximum absorption occurs is called λmax. It is independent of concentration. The drugs are quantitatively analysed by ultraviolet spectroscopic method, it is governed by Beer-Lambert's law, which is represented as

A = abc

Where

A = absorbance

a = absorptivity

b = path length

c = concentration

This relationship exists between the absorbance and the concentration.

Electronic Transitions

Electronic transitions among certain of the energy level can be brought about by the absorption of radiation. Absorbed light causes excitation of electron in a molecule.

There are four important types of transition. They are

σ-σ*

n-σ*

Ï€-Ï€*

n-Ï€*

The energies required for various transitions are in the order

σ-σ*> n-σ* >π-π*> n-π*

A summary of electronic energy levels is shown below

Antibonding (σ*)

Antibonding (Ï€*)

Nonbonding (n)

Bonding (Ï€)

Bonding (σ)

E

When a beam of light passed through species, a part of light absorbed by compound depends upon the concentration and remaining is transmitted. A linear relationship exists between concentrations versus absorbance, which can be used to determine the concentration of unknown substance.

QUANTITATIVE SPECTROPHOTOMETRIC ASSAY OF MEDICINAL SUBSTANCES

1. Use of a calibration graph

Statistical treatment of the calibration data, facilitated by micro computers or pre programmable calculators, provides a more elegant and accurate determination of the relationship between absorbance and concentration than manually constructed graphs. If the absorbance values and concentrations bear a linear relationship, the regression line y = α+βx may be estimated by the method of least squares.

α = (Σy)(Σx2) - (Σx)( Σxy)

N Σx2 - (Σx)2

β = NΣxy-(Σx)( Σy)

NΣx2 - (Σx)2

Where, x = concentration

y = absorbance

N = number of pairs of values

2. Single - point standardization method

The single-point procedure involves the measurement of the absorbance of a sample solution and of a standard solution, of the reference substance. The standard and sample solutions are prepared in a similar manner. Ideally, the concentration of the standard solution should be close to that of the sample solution. The concentration of the substance in the sample is calculated from the proportional relationship that exists between absorbance and concentration.

Ctest=CstdÃ-Astd/Atest

Where C test and C std are the concentrations in the sample and standard solutions respectively, and A test , Astd are the absorbance of the sample and standard solutions respectively. Since sample and standard solutions are measured under identical conditions, this procedure is the preferred method of assay of substances that obey Beer's Law and for which a reference standard of adequate purity is available

3.Difference Spectrophotometry

Difference spectroscopy provides a sensitive method for detecting small changes in the environment of a chromophore or it can be used to demonstrate ionization of a chromophore leading to identification and quantitation of various components in a mixture. The selectivity and accuracy of spectrophotometric analysis of samples containing absorbing interferents may be markedly improved by the technique of difference spectrophotometry. The essential feature of a difference spectrophotometric assay is that the measured value is the difference absorbance (Δ A) between two equimolar solutions of the analyte in different forms which exhibit different spectral characteristics.

The criteria for applying difference spectrophotometry to the assay of a substance in the presence of other absorbing substances are that:

A) Reproducible changes may be induced in the spectrum of the analyte by the addition of one or more reagents.

B) The absorbance of the interfering substances is not altered by the reagents.

The simplest and most commonly employed technique for altering the spectral properties of the analyte properties of the analyte is the adjustment of the pH by means of aqueous solutions of acid, alkali or buffers. The ultraviolet-visible absorption spectra of many substances containing ionisable functional groups e.g. phenols, aromatic carboxylic acids and amines, are dependent on the state of ionization of the functional groups and consequently on the pH of the solution. If the individual absorbances, Aalk and Aacid are proportional to the concentration of the analyte and path length, the Δ A also obeys the Beer-Lambert law and a modified equation may be derived

Δ A = Δ abc

Where Δ a is the difference absorptivity of the substance at the wavelength of measurement.

If one or more other absorbing substances is present in the sample which at the analytical absorbance Ax in the alkaline and acidic solutions, its interference in the spectrophotometric measurement is eliminated

Δ A = (Aalk + Ax) - (Aacid + Ax)

The selectivity of the Δ A procedure depends on the correct choice of the pH values to induce the spectral change of the analyte without altering the absorbance of the interfering components of the sample. The use of 0.1M sodium hydroxide and 0.1M hydrochloric acid to induce the Δ A of the analyte is convenient and satisfactory when the irrelevant absorption arises from pH-insenstive substances. Unwanted absorption from pH-sensitive components of the sample may also be eliminated if the pKa values of the analyte and interferents differ by more than 4.           

HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY

HPTLC is a sophisticated and automated form of TLC. It allows for the analysis of a broad number of compounds both efficiently and cost effectively, it enables the most complicated separations. It is a planar chromatography or flat bed chromatography.

Principle

The principle of separation is adsorption. The mobile phase solvents flow through because of capillary action. The components are separated based on the affinity of the components towards the stationary phase.

Steps involved in HPTLC

Selection of HPTLC plates and sorbents

Layer

Pretreatments

Selection of HPTLC plates and sorbents

Detection of

Spots

Chromatographic development

Application of sample and standard

Scanning and documentation of chromoplate

Selection of HPTLC plates and sorbents

Precoated plates with different support materials and different sorbents are available. In HPTLC, high performance grade of silica in which the particles are small about 5 μm and very uniform in size are used. The high performance silica gel is more efficient and reproducible than conventional grade of silica. Specific differences in the type and distribution of silanol groups for individual sorbents may result in selectivity difference.

Layer Pretreatments

Prior to chromatography it is common practice to prepare the layers for use by any or all of the following steps. Washing, activation, conditioning and equilibration. Newly consigned pre-coated layers are invariably contaminated by the adsorption of materials from the atmosphere. This can result in irregular and drifting densitometric baselines, ghost peaks in the chromatogram and reduced sample detectability in post chromatographic derivatisation reactions.

The problems are easily remedied by pre washing the layers before use. However, plates exposed to high humidity or kept on hand for longtime may have to be activated by placing in a oven at 110-120°C for 30 min prior to sample spotting.

Sample preparation

The sample preparation is not as demanding as for other chromatographic techniques. The sample preparation procedure is to dissolve the dosage form with complete recovery of intact compound of interest and minimum of matrix with a suitable concentration of analytes for direct application on HPTLC plate. For normal phase chromatography using silicagel precoated plates (more than 80-90% of HPTLC analysis is done using silica gel as sorbent) solvent for dissolving the sample should be non-polar and volatile as far as possible. Sample and reference substances should be dissolved in same solvent to ensure comparable distribution at starting zones.

Application of sample

Sample application is the most critical steps for obtaining good resolution for quantification by HPTLC. Usual concentration of sample is in the range of 0.1-1 μg/ml. Concentration above this cause poor separation. The sample, should be completely transferred to the layer. However under no circumstances, the application process should damage the layer as results in unevenly shaped spots. Preferably samples are applied as bands because it ensures better separation because of rectangular area in which the compounds are present on the plate.

Mobile phase optimization

The selection of mobile phase to separate simple mixtures may not be particularly a difficult problem and can be arrived at quite quickly by guided trial and error methods. A solvent of the correct strength for a single development separation will migrate the sample into the Rf range 0.1- 0.9.

For normal separation HPTLC, the stationary phase used is polar and mobile phase in non-polar. In this type of normal phase, hexane is the strength adjusting solvent for weak and moderately polar compounds. In reverse phase HPTLC, the stationary phase is non polar and the mobile phase is polar. In this, water is always the strength adjusting solvent and the solvents used for mobile phase optimisation are restricted to those solvents that are miscible with water.

Pre conditioning (chamber saturation)

When the plate is introduced in to an unsaturated chamber, during the course of development, the solvent evaporates from the plate mainly at the solvent front. Therefore larger quantity of the solvent shall be required for a given distance, hence resulting in increase in Rf values. If the tank is saturated prior to development, solvent vapour soon get uniformly distributed throughout the chamber, hence less solvent shall be required to travel a particular distance resulting in lower Rf values.

Chromatographic development and drying

Ascending, descending, two dimensional horizontal, multiple over run gradient, radial and anti radial are the most common modes of chromatographic development. Rectangular glass chambers, twin trough chambers, v-shaped chamber, circular and anti-circular, v-chambers and automated multiple development chambers are commonly used for carrying out different types of TLC development.

After development, the plate is removed from the chamber and mobile phase is removed as completely and as quickly as possible.

Detection and visualization

One of the most characteristic features of TLC/ HPTLC is the possibility to utilize post chromatographic off-line derivatization. Detection by UV light is the most preferred method as it is non destructive, spots of fluorescent compounds can be seen at 254 nm (short wavelength) or at 366 nm (long wavelength).

Quantification

The chromatographic development should clearly and completely separate all the compounds of interest with no loss by decomposition, evaporation or irreversible adsorption during application or development. Sample and standard as a rule should be chromatographed on the sample plate under similar conditions.

Advantage

It can simultaneously handle several samples even of divergent nature and composition hence, saving a lot of time which is usually not possible with other analytical techniques.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

High performance liquid chromatography is a very sensitive analytical technique most widely used for quantitative and qualitative analysis of pharmaceuticals. The principle advantage of HPLC compared to classical column chromatography are improved resolution of the separated substance, faster separation times and the increased accuracy, precision and sensitivity.

Principle of Separation and its type

There are four types of chromatography in which the mobile phase is a liquid. The mobile phase is pumped through the packed column, under high pressure.

a) Partition chromatography

i) Normal phase chromatography

ii) Reverse phase chromatography

b) Adsorption or liquid solid chromatography

c) Ion exchange chromatography

d) Size exclusion or gel permeation chromatography

Reverse phase mode

Non-polar stationary phase and polar mobile phase is used here.

Mechanism

Retention by interaction of the stationary phase non-polar hydrocarbon chain with non-polar parts and sample molecules.

Reverse phase chromatography

In RP-HPLC the stationary phase is non-polar often a hydrocarbon and the mobile phase is relatively polar such as water, methanol or acetonitrile. In RPC the solutes are eluted in the order of their decreasing polarities. These are prepared by treating the surface of silanol group with an organochlorosilane reagent.

Method development in RP-HPLC

Retention in RP-HPLC

The RPC retention of a compound is determined by its polarity and experimental conditions, mobile phase, column and temperature.

1. Mobile phase effects

Retention time can be preferably adjusted by changing mobile phase composition or solvent strength in RPC. Retention is less for stronger, less polar mobile phases. Solvent strength depends on

Choice of organic solvent or choice of % B.

Concentration of the organic solvent in the mobile phase A: % B where A is water, B is the organic phase and % is volume % v/v.

A retention range of 0.5<K<20 are allowable for sample to be separated using isocratic condition but 1<K<10 is generally preferred.

a) Choice of organic phase

A mobile phase of 100% acetonitrile is a stronger polar solvent, which might result in (K<0.2), so weaker mobile phase is required to retain the compound. By decreasing the percentage of acetonitrile, retention time will increase. If organic phase is decreased by 10%, the K value increases 3 times approximately. Systematic decrease of % B to investigate sample retention is a simple and convenient way to determine the best mobile phase composition for a given sample.

b) Mobile-Phase strength

Mobile phase strength in RPC depends upon both % B and the type of organic solvent. RPC solvent strength varies as water (weakest) < methanol < acetonitrile < ethanol < tetrahydrofuran < propanol < methylene chloride (strongest). Solvent strength increases as solvent polarity decreases. Any of the above solvents can be used with water for RPC, except methylene chloride since it is not water miscible. Acetonitrile is the best initial choice of organic solvent for the mobile phase. The next best organic solvent is methanol followed by tetrahydrofuran.

2. Selectivity in RP-HPLC

Three main variables can be used in RPC to change selectivity (α) for neutral samples like mobile phase composition, column type and temperature. Overall sample retention acceptable is (0.5< K< 20).

a) Solvent-strength selectivity

The best sample resolution will occur for a % B value, where both pairs have the same resolution peak spacing can be explored while % B is varied for optimum sample retention (0.5< K< 20).

b) Solvent type selectivity

A change in organic solvent type is often used to change peak spacing and improve resolution. The selection of RPC solvents for this purpose is guided by solvent properties that are believed to affect selectivity, acidity, basicity and dipolarity. Only a slight increase (2 to 5 %) in the selectivity (α value) for a critical band pair may be necessary to achieve acceptable resolution. Changing solvent type in RPC is usually the most effective procedure to alter selectivity and achieve the separation of multicomponent neutral samples.

c) Column type selectivity

A change in column type can produce useful changes in selectivity and over all sample retention. Retention is greater (run time longer) on the stronger (C8 and phenyl column) vs. the weaker cyano column. A change of the column is usually less useful than a change in mobile phase type hence this should be tried only after the use of solvent strength or solvent type selectivity has failed. Usually a C8 or C18 column should be tried first followed by a cyano, then by a phenyl column. Column padings bonded with cyclodextrin (CD) are useful in separation of enantiomeric isomers.

d) Temperature selectivity

Values of K decreases at higher temperature for the RPC separation of neutral compounds. This is less effective for non-ionic compounds as a mean of altering selectivity for improved separation. As the temperature is increased, the relative retention of the polar compounds decreases more rapidly than for the non-polar compounds.

FUNDAMENTALS OF SEPARATION

Column efficiency (N)

Column efficiency is called as number of theoretical plates. It measures that the band spreading number of theoretical plate is higher. If it is higher it indicates good column and system performance.

Column performance can be defined on terms of values of N.

N = 16(tR/w)2 or 3500 L (cm)/ dp (μm)

Plate height, H = N /L where L = length

Capacity factor (K')

It is the measure of how well the sample molecule is retained by the column during an isocratic separation. It is affected by solvent composition, separation, aging and temperature of separation.

tR = band retention time

t0 = column dead volume

Resolution

The quality of separation is usually measured by resolution R, of adjacent bands.

t1 and t2 are retention times of the first and second adjacent bands. w1 and w2 are baseline bandwidths.

Asymmetry

The asymmetry is a tool for quickly determining how much if any, of an eluting peak profile deviates in shape from a normal distribution. The subscript 'x' refers to the percentage of peak height at which the asymmetry is determined.

E.g.: A10 (determined at 10% peak height)

The equation for determining peak asymmetry is

Ax = b/a,

Where,

'b' = the distance between the perpendicular connecting the baseline to peak maximum and the latest eluting portion of the curve.

'a' = the distance between the perpendicular connecting the baseline to the peak maximum and the earliest eluting portion of the curve.

Selectivity

It measures relative retention of two components. Selectivity is the function of chromatographic surface (column), melting point and temperature.

ANALYTICAL PARAMETERS FOR VALIDATION

Validation may be defined as a process involving confirmation or establishing by laboratory studies that a method/ system/ analyst gives accurate and reproducible result for intended analytical application in a proven and established range.

Validation parameters

The parameters for method validation as defined by the ICH guidelines are summarized below.

Selectivity (specificity)

Ability of the developed method to detect analyte quantitatively in the presence of other components, which are expected to be present in the sample matrix or other related substances. Results are expressed as resolution. If the expected impurities or related substances are available, they should be analysed along with the analyte or sample to check the system suitability, retention factor, tailing factor, resolution etc.

Linearity

It is the ability of the method to elicit test result that is directly proportional to analyte concentration within a given range. It is generally reported as variance of slope of regression line. It is determined by series of three to six injections of five of more standards.

Range

It is the interval between the upper and lower levels of analyte, which is studied. The range is normally expressed in the same units as the test results obtained by the analytical method. The ICH guidelines specify a minimum of five concentration levels.

Precision

It is a measure of degree of repeatability of an analytical method under normal operation and it is normally expressed as % of relative standard deviation (% RSD). This involves

Repeatability

Reproducibility

Intermediate precision

% RSD = 100 S/X

Where, S = Standard deviation

X = Mean

It is determined at three levels.

a) Repeatability

Precision of the method when repeated by the same analysts, same test method and under same set of laboratory conditions (reagent, equipments), within a short interval of time, the only difference being the sample.

b) Reproducibility

When the subject method is carried out by different analysts in different laboratories using different equipments, reagents and laboratory settings and on different days of variability of analytical results as function of analyst, day to day, laboratory to laboratory, equipment to equipment etc., using the samples from same homogenous batch.

c) Intermediate precision

It is determined by comparing the results of a method within the same laboratory but different days, analysts, equipments and reagents.

Accuracy

Defined as the closeness of agreement between the actual (true) value and mean analytical value obtained by applying the test method a number of times. Accuracy is acceptable if the difference between the true value and mean measured value does not exceed the RSD values obtained for repeatability of the method.

One can design experiments for recovery of known or spiked samples in presence of expected matrix, keeping the matrix constant. Accuracy can also be determined by comparing the results with those obtained using an alternative method which has already been validated.

Limit of Detection (LOD)

It is defined as the lowest concentration of an analyte in a sample that can be detected but not quantified. LOD is expressed as a concentration at a specified signal to noise ratio. The LOD will not only depend on the procedure of analysis but also on the type of instrument.

In chromatography, detection limit is the injected amount that results in a peak with a height at least twice or thrice as high as baseline noise level.

S/N = 2/1 or 3/1

Limit of Quantification (LOQ)

It is defined as lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy and reliability by a given method under stated experimental conditions. The procedure usually followed is to analyze samples containing decreasing known quantity of the analyte and determine the lowest level at which acceptable level of accuracy is attained.

LOQ is expressed as a concentration at a specified signal to noise ratio. In chromatography, limit of quantification is the injected amount that results in a peak with a height, ten times as high as base line noise level.

S/N = 10/1

Ruggedness

Degree of reproducibility of test results obtained by analyzing the same sample under variety of normal test conditions such as different analysts, instruments, days, reagents, column and TLC plates.

Robustness

It is the measure of the capacity of the analytical method to remain unaffected by small but deliberate variation in procedure. It provides an indication about variability of the method during normal laboratory conditions.

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