Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Selection and calibration of analytical method & calibration methods

The accuracy of a measurement system is the degree of closeness of measurements of a quantity to the true value.
The precision  of a measurement system, also called reproducibility or repeatability, is the degree to which repeated measurements under unchanged conditions show the same results.

The sensitivity of a clinical test refers to the ability of the test to correctly identify those patients with the disease.

A test with 100% sensitivity correctly identifies all patients with the disease.

A test with 80% sensitivity detects 80% of patients with the disease (true positives) but 20% with the disease go undetected (false negatives).

The specificity of a clinical test refers to the ability of the test to correctly identify those patients without the disease.

Therefore, a test with 100% specificity correctly identifies all patients without the disease.

A test with 80% specificity correctly reports 80% of patients without the disease as test negative (true negatives) but 20% patients without the disease are incorrectly identified as test positive (false positives).

The specificity of a clinical test refers to the ability of the test to correctly identify those patients without the disease.

Therefore, a test with 100% specificity correctly identifies all patients without the disease.

A test with 80% specificity correctly reports 80% of patients without the disease as test negative (true negatives) but 20% patients without the disease are incorrectly identified as test positive (false positives).

  • Be the first to comment

Selection and calibration of analytical method & calibration methods

  1. 1. TapeshwarYadav (Lecturer) BMLT, DNHE, M.Sc. Medical Biochemistry Selection of analytical method
  2. 2. Selection criteria accuracy,  precision, sensitivity, Detection limit, selectivity, Linearity and Range •robustness, •ruggedness, •scale of operation, • equipment, •Time • cost.
  3. 3. Accuracy  The accuracy of a measurement system is the degree of closeness of measurements of a quantity to the true value
  4. 4. Precision The precision of a measurement system, also called reproducibility or repeatability, is the degree to which repeated measurements under unchanged conditions show the same results.
  5. 5. Sensitivity The sensitivity of a clinical test refers to the ability of the test to correctly identify those patients with the disease. A test with 100% sensitivity correctly identifies all patients with the disease. A test with 80% sensitivity detects 80% of patients with the disease (true positives) but 20% with the disease go undetected (false negatives).
  6. 6. Specificity The specificity of a clinical test refers to the ability of the test to correctly identify those patients without the disease. Therefore, a test with 100% specificity correctly identifies all patients without the disease.  A test with 80% specificity correctly reports 80% of patients without the disease as test negative (true negatives) but 20% patients without the disease are incorrectly identified as test positive (false positives).
  7. 7. Sensitivity and Specificity
  8. 8. Detection Limit Limit of Detection (LOD) or detection limit, is the lowest concentration level that can be determined to be statistically different from a blank (99% confidence). The LOD is typically determined to be in the region where the signal to noise ratio is greater than 5. Limits of detection are matrix, method, and analyte specific
  9. 9. Instrument Detection Limit (IDL) Instrument Detection Limit (IDL) is the concentration equivalent to a signal, due to the analyte of interest, which is the smallest signal that can be distinguished from background noise by a particular instrument.
  10. 10. Method Detection Limit (MDL) Method Detection Limit (MDL) is the minimum concentration of a substance that can be measured and reported with 99% confidence that the analyte concentration is greater than zero. Determined from analysis of a sample in a given matrix containing the analyte
  11. 11. Selectivity criterion of an analytical method is its capability to deliver signals that are free from interferences and give "true results". Selectivity of a method refers to the extent to which it can determine particular analyte(s) in a complex mixture without interference from other components in the mixture Specificity is the ultimate of Selectivity
  12. 12. Robustness  Robustness is the capacity of a method to remain unaffected by small deliberate variations in method parameters..
  13. 13. Ruggedness Ruggedness is defined as the degree of reproducibility of the test results obtained under a variety of normal test conditions, such as different laboratories, different analysts, different instruments, different lots of reagents, different elapsed assay times, different assay temperatures, different days, etc
  14. 14. Linearity and Range Linearity is the ability of the method to elicit test results that are directly proportional to analyte concentration with in a given range. Traditionally linearity was regarded as a desirable property of methods as only linear curves could be easily interpreted.. Range is the interval between lower and upper levels of analyte that is demonstrated to be determined with the stated precision and accuracy using the method as written.
  15. 15. Contd… Scale of Operation  Equipment Time  Cost.
  16. 16. Calibration of InstrumentalCalibration of Instrumental MethodsMethods
  17. 17. Introduction Important part of all analytical procedures is the calibration and standardization process. Calibration determines the relationship between the analytical response and the analyte concentration. This is determined by the use of chemical standards.
  18. 18. Almost all analytical methods require some type of calibration with chemical standards. Exception Gravimetric methods and some coulometric methods .
  19. 19. Comparison with Standards Direct Comparison Some analytical procedures involve comparing a property of the analyte (or the product of a reaction with the analyte) with standards such that the property being tested matches or nearly matches that of the standard. Early colorimetric methods, the color produced as the result of a chemical reaction of the analyte was compared with the color produced by reaction of standards.
  20. 20. Direct Comparison
  21. 21. Direct Comparison If the concentration of the standard is varied by dilution, it is possible to obtain a fairly exact color match. The concentration of the analyte will be equal to the concentration of the standard after dilution. This procedure is called a null comparison or isomation method.'
  22. 22. TitrationsTitrations Titrations are among the most accurate of all analytical procedures. The analyte reacts with a standardized reagent (the titrant) in a reaction of known stoichiometry. Usually the amount of titrant is varied until chemical equivalence is reached, as indicated by the color change of a chemical indicator or by the change in an instrument response. The amount of the standardized reagent needed to achieve chemical equivalence can then be related to the amount of analyte present. The titration is thus a type of “chemical comparison.”
  23. 23. External Standard Calibration An external standard is prepared separately from the sample. External standards are used to calibrate instruments and procedures when there are no interference effects from matrix components in the analyte solution.
  24. 24. Series of such external standards containing the analyte in known concentrations is prepared. Ideally, three or more such solutions are used in the calibration process. However, in some routine analyses, two-point calibrations can be reliable.
  25. 25. External Standard Calibration… Calibration is accomplished by obtaining the response signal (absorbance, peak height, peak area) as a function of the known analyte concentration. A calibration curve is prepared by plotting the data or by fitting them to a suitable mathematical equation,
  26. 26. The next step is the prediction step, where the response signal is obtained for the sample and used to predict the unknown analyte concentration,
  27. 27. The Least-Squares Method The method of least squares is based on two assumptions. The first is that there is actually a linear relationship between the measured response y and the standard analyte concentration x Y= mx+b where b is the y intercept (the value of y when x is zero)  m is the slope of the line
  28. 28. In cases where the data do not Fit a linear mode nonlinear regression methods are available.
  29. 29. In a determination, the raw response from the instrument is not used. Instead, the raw analytical response is corrected by measuring a blank.  An ideal blank is identical to the sample but without the analyte.
  30. 30. In practice, with complex samples, it is too time-consuming or impossible to prepare an ideal blank and a compromise must be made. Most often a real blank is either a solvent blank, containing the same solvent in which the sample is dissolved, or a reagent blank, containing the solvent plus all the reagents used in sample preparation.
  31. 31. Errors in calibration Systematic errors can occur during the calibration process. For example, if the standards are prepared incorrectly ,an error will occur Concentration of the standards can change because of decomposition, volatilization or adsorption onto container walls. Contamination of the standards can also result in higher analyte concentrations than expected
  32. 32. Errors in calibration Random errors can also influence the accuracy of results obtained from calibration curve
  33. 33. Errors in External-Standard CalibrationErrors in External-Standard Calibration Matrix effects, due to extraneous species in the sample that are not present standards or blank, can cause the same analyte concentrations  Differences in experimental variables at the times at which blank, sample, and standard are measurement. Even when the basic assumption is valid, errors can still occur because of contamination during the sampling or sample preparation steps.
  34. 34. Multivariate CalibrationMultivariate Calibration Univariate calibration procedure measures only one response used per sample. The process of relating multiple instrument responses to an analyte or a mixture of analytes is known as multivariate calibration. Multivariate calibration methods have become quite popular in recent years as new instruments become available that produce multidimensional responses
  35. 35. Absorbance of several samples at multiple wavelengths, mass spectrum chromatographically separated components, etc.). They can also be used to detect the presence of interferences that would not be identified in a univariate calibration.
  36. 36. Standard-Addition MethodsStandard-Addition Methods Particularly useful for analysing complex samples in which the likelihood of matrix effects is substantial. One of the most common forms involves adding one or more increments of a standard solution to sample aliquots containing identical volumes. This process is often called spiking the sample. Each solution is then diluted to a fixed volume before measurement.
  37. 37. Internal Standard methodInternal Standard method An internal standard is a substance that is added in a constant amount to all samples, blanks, and calibration standards in an analysis. Calibration then involves plotting the ratio of the analyte signal to the internal-standard signal as a function of the analyte concentration of the standards.
  38. 38. Internal Standard methodInternal Standard method The internal standard should provide a signal that is similar to the analyte signal in most ways but sufficiently different so that the two signals are distinguishable by the instrument. The internal standard must be known to be absent from the sample matrix so that the only source of the standard is the added amount.

×