Analytical chemistry1 is the knowledge to evaluate morphologies, compositions, and quantities of analytical targets. These analytical consequences have played significant roles from the perceptive of basic science to a diversity of practical applications, such as biomedical applications, environmental monitoring, quality control of industrial manufacturing, and forensic science, to name a few. Analytical chemistry is concerned with chemical characterization of matter, both qualitative and quantitative.
Pharmaceutical Analysis plays a extremely important role in quality control of pharmaceuticals through a rigid check on raw materials used in manufacturing of formulations and on finished products. It also plays a main role in building up the quality products through in development quality control. Pharmaceutical analysis is the application of principles of analytical chemistry to drug analysis. The analytical chemistry may be defined as the art of developing sensitive, relative and accurate methods for shaping the composition of materials in terms of elements or compounds which they enclose.
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Introduction1: Chromatography (from Greek: chroma, colour and: "grafein" to write) is the communal word for a family of laboratory experiments for the partition of mixtures. Chromatography encompasses a diverse and important group of methods that allow the separation, identification, and determination of closely related components of complex mixtures.
Chromatography will be of two types; preparative or analytical. Preparative chromatography used for auxiliary use i.e, a form of purification. Analytical chromatography usually operates with trace amounts of material and used to determine the relative proportions of analytes in a mixture.
High-performance liquid chromatography (HPLC)
In the field of analytical chemistry high performance liquid chromatography (HPLC) is considered by many to be most exciting and dynamic technique of past decade. Since its advent in 1969, tremendous improvements have been realized in pumping system, sample introduction modes, column design and detector to make it a rapid, accurate, and precise technique for analytical determination of compounds. The numbers of mobile phases in HPLC are infinite and thus separation possibilities are limited only to the analyst's imagination.
High performance liquid chromatography is a suitable separation technique used for wide types of samples, with outstanding resolving power, speed and nano molecular detection levels. It is right now used in pharmaceutical research and developments in the subsequent ways:
To assay active ingredients, impurities, degradation products and in dissolution assays
To purify synthetic or natural products
To characterize metabolites
In pharmacodynamics and pharmacokinetic studies
Chromatography encompasses a varied group of methods that are utilized for the separation of narrowly related components of mixtures. In all chromatographic separations, the sample is transported within the mobile phases, which may be a gas (GC), a liquid (LC), or a supercritical fluid (SFC). In column chromatography, the stationary phase is enclosed within a narrow tube through which the mobile phase is forced by gravity or under pressure. The components of the mixture to be analyzed distribute themselves between the mobile phase and stationary phase in varying proportions. Compounds that interrelate strongly with the stationary phase travel very slowly with the mobile phase; in contrast, compounds that are weakly retained by the packing material travel rapidly with the mobile phase. As a significance of the differences in mobility between the individual components of a mixture, the sample components are separated into distinct bands (or zones) that emerge from the column at precise 'retention times'. These bands may be identified qualitatively and /or further analyzed quantitatively by means of an appropriate detector.
The typical HPLC separation is based on the discriminating distribution of analytes between a liquid mobile phase and an immiscible stationary phase. The sample is first introduced by means of an injection port into the mobile phase stream that is delivered by a high-pressure pump. Subsequently, the components of this sample mixture are separated on the column, a process monitored with a flow-through detector as the isolated components emerge from the column.
A further modification to HPLC has been to contrast the mobile phase composition during the analysis; this is called as gradient elution.
Schematic diagram of HPLC instrument
Types of pumps in HPLC4, 5
Pumps are most important component of HPLC and their performance directly affects the detector reproducibility and detector's sensitivity. Their function is to force the liquid (mobile phase) through the column of finely packed particles
Always on Time
Marked to Standard
Syringe pump (screw driven)
- Single piston reciprocating pump
- Dual piston reciprocating pump
- Reciprocating diaphragm pump
- Direct pressure pump
- Amplifier pump
Since sample valves come between the pump and the column it follows that HPLC sample valves must also tolerate pressures up to 10,000 psi For analytical HPLC, the sample volume should be selectable from sub- micro litre to a few micro litres, whereas in preparative HPLC the sample volume may be even greater than 10 ml.
HPLC columns are packed with very fine particles (usually a few microns in diameter). The very fine particles are required to attain the low dispersion that give the high plate counts expected of modern HPLC. Plate counts in excess of 25,000 plates per column are possible with modern columns because of the dispersion associated with injection valves, detectors, data acquisition systems and the dispersion due to the higher molecular weight of real samples as opposed to the common test samples. The column will retain those substances that interact more strongly with the stationary phase than those that interact more strongly with the mobile phase
Different types of columns are used. They are
Narrow bore column
Analytical column variables are as follows
Length (10-30 cm)
ID (4-10 mm)
Packing (many kinds)
Particles sizes (3-10 Âµm)
Most common columns 25 cm x 4.6 i.d with 5Âµ particles
Detector is the eye of LC system and measures the compounds after the separation on the column. Before the first sample is injected, during method development the chromatographer must make sure that the detector so selected is skilled of responding to changes in the concentration of all the components in the sample with sufficient sensitivity even to measure trace substances.
There are basically two types of detectors.
Bulk property detectors
Solute property detectors
The Bulk property detectors function on some bulk property of the eluent, such as refractive index and are not appropriate for gradient elution and are typically less sensitive than solute property detectors. The solute property detectors carry out by measuring some type of physical or chemical property that is specific to the solute only [Sethi,2001].
Methods in Chromatography [Willard, 1986, Sethi, 2001]
Normal phase chromatography
Reserved phase chromatography
Ion exchange chromatography
Affinity phase chromatography
Hydrophobic Interaction chromatography (HIC)
- Liquid liquid partition chromatography
Size exclusion chromatography (SEC)
- Gel permeation chromatography
- Gel chromatography
- Gel Filtration
Isocratic flow and gradient elution
By consider to the mobile phase, a composition of the mobile phase that leftovers stable throughout the procedure is termed isocratic.
In differentiate to this is the so known as "gradient elution", which is a separation where the mobile phase changes its composition during a separation process. One eg; is a gradient in 20 min starting from 10Â % Methanol and ending up with 30Â % Methanol. Such a gradient be able to be increasing or decreasing.
Quantification involves the measurement of peak height or peak area. To determine the concentration of a compound, the peak area or height is plotted Vs the concentration of the substance. For well resolved peaks, both peak height and area are proportional to the concentration. Four different calibration methods used in quantitative analysis are,
Normalized peak area
External standard addition method
Internal standard addition method and
Standard addition method
Normalized Peak Area
The area present of any individual peak is referred to as the normalized peak area. The performance of normalized peak area is essentially not a calibration method, since there is no similarity to known amounts for any peak in the chromatogram.
External Standard Calibration
The most universal method for determining the concentration of an unknown sample is to construct a calibration plot using external standards. Standards are prepared at known concentrations. A rigid volume of each standard solution is injected and analyzed, and the peak responses are plotted Vs concentration. The standard solutions are referred to as external standards, since they are prepared and analyzed in separate chromatograms from those of the unknown samples. Unknown samples are then prepared, injected and analyzed in exactly the same manner.
Internal Standard Calibration
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The internal standard is a different compound form the analyte, but solitary that is well resolved in the separation. The internal standard can recompense for changes in concentration to instrumental variations. With the internal standard method, a calibration plot is produced by preparing and analyzing calibration solutions containing different concentration of the compound of interest with a fixed concentration of the internal added.
The Internal standard must complies the following requirements: [Snyder et al., 1997, Sharma B.K., 1980]
Well resolved from the compound of Interest and other peaks.
Should not be in the original sample
Similar retention (k) to the analyte.
Does not have to be chemically similar to analyte.
Should ape the analyte in any sample preparation steps.
Stable and unreactive with sample or mobile phase.
Commercially accessible in high purity.
It must be separated from all compounds of significance in the separation.
Should have similar detector rejoinder to the analyte for the concentration used
It must be separated from all compounds of significance in the separation.
Method of Standard Addition
A calibration standard ideally should be prepared in a blank matrix of drug formulation components without the drug substance or an animal exclusive of added compound usually can be used for standard calibration solutions. The method of standard addition is most frequently used in trace analysis. In this progress, different weights of analyte(s) are added to the sample matrix, which initially contains an unknown concentration of the analyte. Extrapolation of a plot of response found for the standard addition calibration concentration to zero concentration defines the original concentration in the unspiked sample.
STEPS INVOLVED IN METHOD DEVELOPMENT OF HPLC
HPLC Method Development for the analysis of mixtures of substances is a task that usually requires much expertise. It is also extremely time-consuming. In spite of advances in chromatographic theory, HPLC Method Development is still based mainly on "trial and error". Consequently, many attempts have been made to use computer programs to facilitate this process.
Prediction of starting conditions
Selection of column
Selection of wavelength
Selection of mobile phase
The empirical approach from analyte structures
Chemical nature of the drug
Optimization of linear and multi step conditions
Performing the trials based on the result obtained
Introduction TO VALIDATION6, 7, 8, 9
Method validation is the process to confirm that the analytical procedure employed for a specific test is suitable for its intended use. Methods need to be validated or revalidated.
â€¢ Before their introduction into routine use
â€¢ Whenever the conditions change for which the method has been validated, e.g., instrument with different characteristics
â€¢ Whenever the method is changed, and the change is outside the original scope of the method.
Method validation is completed to ensure that an analytical methodology is accurate, specific, reproducible and rugged over the specified range that an analyte will be analyzed. Method validation provides an assurance of reliability during normal use, and is sometime referred to as "the process of providing documented evidence that the method does what it is intended to do." Regulated laboratories must perform method validation in order to be in compliance with FDA regulations.
For method validation, these specifications are listed in USP Chapter <1225>, and can be referred to as the "Eight Steps of Method Validation," as shown in figure below. These terms are referred to as "analytical performance parameters", or sometimes as "analytical figures of merit." Most of these terms are familiar and are used daily in the laboratory. However some may mean different things to different people. Therefore, in order to continue the discussion of method validation, it is necessary to have a complete understanding of the terminology and definitions.
The USP Eight Steps of Method Validation
In response to this situation, one of the first harmonization projects taken up by the ICH was the development of a guideline on the "Validation of Analytical Methods: Definitions and Terminology." ICH divided the "validation characteristics" somewhat differently, as outlined in Figure below
ICH Method Validation Parameters
The developed methods were validated by following steps
â€¢ Limit of quantitation
â€¢ Limit of detection
â€¢ Linearity of range
â€¢ Ruggedness and
It is defined as closeness of agreement between the actual (true) value and mean analytical value obtained by applying a test method number of times. Accuracy of an analytical method is determined by systematic error involved. The 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.
The parameter provides information about the recovery of the drug from sample and effect of matrix, as recoveries are likely to be excessive as well as deficient.
The precision of an analytical method is the closeness of agreement among individual test results when the method is apply repetitively to multiple sampling of homogenous sample.
Precision is the measure of the degree of repeatability of an analytical method under normal operation and is normally expressed as the variance, percent relative standard deviation for a statistically significant number of samples.
Repeatability is the results of the method operating over a short time interval under the same conditions. It is also called as an inter-assay precision. We can be determined from a least of nine determinations cover the precise range of the procedure (for example, three levels, three repetitions each) or from a minimum of six determinations at 100% of the test or target concentration.
Intermediate precision is the results from within lab variations due to random events such as different days, analysts, equipment, etc. In determining intermediate precision, experimental design should be employed so that the effects (if any) of the individual variables can be monitored.
Reproducibility refers to the results of collaborative studies between laboratories.
Documentation in support of precision studies should include the standard deviation, relative standard deviation, coefficient of variation, and the confidence interval.
It is defined as the capability of an analytical method to evaluate unequivocally the analyte of significance in the presence of components that may be expected to be present, such as impurities, degradation products and matrix components.
Limit of Detection (LOD):
The limit of detection is a characteristic of limit test. It is the lowest concentration of analyte in a sample that can be detected but not necessarily quantified. The detection limit is usually expressed as the concentration at a specified signal to noice ratio.
Limit of Quantization:
The Limit of Quantization (LOQ) is the distinctive of quantitative assays. It is the lowest amount of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental condition. The quantitation limit is expressed as the concentration of analyte that would yield signal to noice ratio.
Linearity and Range:
The linearity of an analytical method is its ability to elict test results, which are directly proportional to the concentration of analyte in sample with in a given range.
The range of an analytical method ia an interval between the upper and lower levels of analyte (including these levels) that have been demonstrated to be determined with a suitable level of precission, accuracy and linearity .
Robustness is the capacity of a method to remain unaffected by small deliberate variations in method parameters. The robustness of a method is evaluated by varying method parameters such as percent organic, pH, ionic strength, temperature, etc., and determining the effect (if any) on the results of the method. As documented in the ICH guidelines, robustness should be considered early in the development of a method. In addition, if the results of a method or other measurements are susceptible to variations in method parameters, these parameters should be adequately controlled and a precautionary statement included in the method documentation.
System Suitability Test (SST)
SST is commonly used to verify resolution, column efficiency, and repeatability of the chromatographic system to ensure its adequacy for a particular analysis. According to the United States pharmacopoeia (USP) and the International Conference on Harmonization (ICH), SST is an integral part of many analytical procedure.
Primary SST parameters are most important as they indicate system specificity, precision and column stability. Other parameter include capacity factor (K) and signal to noise ratio (S/N) for impurity peaks. The purpose of system suitability test is to ensure that the complete testing system (including instrument, reagents, column, and analyst) is suitable for intended application.
SYSTEM SUITABILITY PARAMETERS FOR HPLC9
Retention time (RT)
Retention time is time of elution of peak maximum after injection of sample.
Column Efficiency (N)
Solutes are placed on an HPLC column in a narrow band
Each solute band spreads as it moves through the column due to diffusion and mass transfer affects
The later eluting bands will spread more
Peak shape follow a Gaussian distribution
The sharpness of a chromatographic peak is an indication of the quality of the chromatographic column.
Peak sharpness is determined by measurement of the peak width
Peak width is dependent on flow rate so measurement of the width alone is not enough
A good measure of column efficiency is.
n = 16 () 2 or n = 5.54 ( ) 2
w = width of the peak at its base, obtained by extrapolating the relatively straight sides of the peak to the baseline.
w1/2 = width of the peak at half height, obtained directly by electronic integrators.
The value of 'N' depends upon the substance being chromatographed as well as the operating conditions such as mobile phase, temperature etc
It is function of column efficiency, and is specified to ensure that closely eluting compounds are resolved from each other to establish the general resolving power of system.
The separation of two components in a mixture the resolution is determined by the equation,
Resolution between two peaks.
Where, tR(2) and tR(1) are the retention time of second and first compound respectively, where as W2 and W1 are the corresponding widths at the base of peaks obtained by extrapolating straight side of peaks to the base lines.
Where, electronic integrator is used, it may be convenient to determine the resolution by equation
Where, W h/2 is peak at the half height, obtained directly by electronic integrator.
Peak Asymmetric factor or Tailing (As)
A properly packed HPLC column will give symmetrical or gaussian peak shapes. Changes in either the physical or chemical integrity of the column bed can lead to peak tailing.
Where W0.05 is the width of peak at 5% height and f is the distance from the peak maximum to the leading edge of the peak height from the baseline.
Tailing can be caused by:
Column voids, channels, or extra-column dead volume (affects early eluting peaks most)
Stripping of the bonded phase (affects late eluting peaks most)
Separation Factor (Î±ï€ )
The separation factor, also referred to as column selectivity, is affected by changes in the chemistry of the chromatographic method such as:
A change in the choice of solvents for the mobile phase
A change in the packing material in the column
Because we are usually dealing with samples that contain more than one sample component, a term describing the separation of peaks is needed.
The separation factor describes the relative position of two peak maxima
It is equal to the ratio of the time each component spends on the packing material
Î± = ï€
This equation is more often seen as the ratio of the capacity factors
Î± = ï€
Capacity Factor (k')
Capacity factor is the ratio of the reduced retention volume to the dead volume. Capacity factor, k', is defined as the ratio of the number of molecules of solute in the stationary phase to the number of molecules of the same in the mobile phase. Capacity factor is a measure of how well the sample molecule is retained by a column during an isocratic separation. The ideal value of k' ranges from 2-10. Capacity factor can be determined by using the formula,
Where, tR = retention volume at the apex of the peak (solute) and
t0 = void volume of the system.
A change in capacity factor signifies at least one of the following:
A change in the strength of the mobile phase
- can be caused by evaporation of one of the mobile phase components
Contamination of binding sites
- Strongly bound sample components can reduce the number of binding sites available and lower the column's capacity to retain other solutes
- The use of guard columns will minimize this problem
- Column regeneration can help restore column capacity
loss of binding sites
-harsh mobile phases (low or high pH) can cause loss of bonded phase
or loss of end capping
-this often causes a change in selectivity.
Due toÂ the nature of the chromatographic equipment a dilution of the injected sample solution occurs when the sample molecules migrates through the column. As a consequence the zone containing the sample molecules broadens continually during its passage through the column. The detector will register a peak with a certain width. The measureÂ for the peak widthÂ is the height of a theoretical plate. Â
System Suitability Parameters and Recommendations
Capacity Factor (k') The peak should be well-resolved from other peaks and the void volume, generally k'>2.0
RepeatabilityÂ Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â RSD </= 1% for N >/= 5 is desirable.
Relative retentionÂ Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Not essential as long as the resolution is stated.
Resolution (Rs)Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Rs of > 2 between the peak of interest and the closest eluting
potential interferent (impurity, excipient, degradation product, internal standard, etc.
Tailing Factor (T)Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â T of </= 2
Theoretical Plates (N)Â Â Â Â Â Â Â Â Â Â Â Â Â Â In general should be > 2000
STATISTICAL PARAMETERS [Kamboj, 2003]
Once a linear relationship has been shown to have a high probability by the value of the correlation coefficient 'r', then the best straight line through the data points has to be estimated. This can often be done be done by visual inspection of the calibration graph, but in many cases it is far more sensible to evaluate the best straight line by linear regression (the method of least squares).
The equation of straight line is
y = mx + c
Where, y the dependent variable is plotted as result of changing x, the independent variable.
To obtain the regression line 'y on x' the slope 'm' of the line and the intercept 'c' on the y axis are given by the following equation.
m = and c =
To establish whether there is a linear relationship between two variables x1 and y1, use Pearson's correlation coefficient r.
Where n is the number of data points.
The value of r must lie between +1 and -1, the nearer it is to +1, the greater the probability that a definite linear relationship exists between the variables x and y, values close to +1 indicate positive correlation and values close to -1 indicate negative correlation values of 'r' that tend towards zero indicate that x and y are not linearly related (they made be related in a non-linear fashion).
It is commonly used in statistics as a measure of precision statistics as a measure of precision and is more meaningful than is the average deviation. It may be thought of as a root-mean-square deviation of values from their average and is expressed mathematically as
S is standard deviation.
If N is large (50 or more) then of course it is immaterial whether the term in the denomination is N -1 or N
Î£ = sum
= Mean or arithmetic average
= deviation of a value from the mean
N = Number of observations
Percentage relative standard deviation (%RSD)
It is also known as coefficient of variation CV. It is defined as the standard deviation (S.D) expressed as the percentage of mean.
S.D = standard deviation,
= Mean or arithmetic average.
The variance is defined as S2 and is more important in statistics than S itself. However, the latter is much more commonly used with chemical data.
Standard error of mean (S.E.)
Standard error of mean can be defined as the value obtained by division of standard deviation by square root of number of observations. It is mathematically expressed as
S.D = Standard deviation
n = number of observations.