2.

DATA ANALYSIS (Part 2)

CALIBRATION GRAPHS & VALIDATION

Analytical data can be obtained by observing the RESPONSE of an assay Response is the effect of an analyte in an assay, assay , that may be observed by the following: Any change from normal that can be quantified by measurement Area under a curve Height of chromatographic peak Measure of transmittance, absorbance or reflectance of light
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CALIBRATION GRAPHS

Quantification Using a Standards

1. EXTERNAL STANDARD CALIBRATION METHOD Works well when there are no effects of sample matrix on the signal or sample recovery during preparation The standard and the sample are analyzed separately under identical conditions The blank must be subtracted from the readings obtained Dilutions of the sample is necessary if the response do not fit into calibration graph
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Used to find the quantitative relation between

two variables

The y-vertical axis is instrument response and the

x-horizontal axis is concentration Graphs are normally linear (however not all points are on the straight line due to random error) Regression analysis is done on to check for 1.0) ) linearity of data (R2 value 1.0 Types of Calibration graph: External Calibration Standard Addition Calibration Internal Standard Calibration

General Procedure

Prepare standard stock solution of analyte Prepare standards by serial dilutions from
A bsorbance

Sample standard calibration graph

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0

standard stock Prepare sample and blank Test for the analyte in the serial solutions and the unknown sample under identical conditions

Obtain the response at each concentration Graph out the standard calibration graph (using Excel) Generate linear equation and determine x

y = 0.009x + 0.0044 2 R = 0.9991

y = mx + b
y is the response x is the concentration m is slope

1 ppm

2 ppm Blank

3 ppm

4 ppm Sample

5 ppm

50 100 Concentration

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Y-axis is the dependent variable X-axis is the independent variable 5

R2 1.0 indicates the linearity of data

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The cutoff of a standard calibration graph is -

dependent on the assay Example: In this assay, the linear equation is only valid up to absorbance of 0.5 units, thus the samples with readings >0.5 must be diluted (extrapolations are not allowed)
0.6 0.5 0.4 0.3 0.2 0.1 0 0 2 4 8 16 62 64 132 264

Example 1: Determine the concentration of Zn in water sample Zn (ppm) 0.00 1.00 2.00 3.00 4.00 5.00 Sample? Signal 0.00 0.06 0.13 0.21 0.25 0.29 0.22 How to prepare serial dilutions from a 1000ppm stock solution to 100pm & to the standards? The blank must be subtracted from the readings obtained Increase in analytical signal is related to the quantity of the analyte 8

Absorbance

Concentration

EXAMPLE 2 Determine the concentration of analyte in the sample given the data in columns 1 and 2 Solution: Solution : Since the reading obtained for the blank is not zero, subtract the blank intensity from all original data Concentration (ppm) 0.00 (blank) 0.20 0.50 1.00 1.50 2.00 2.50 Sample Original Intensity 4.5 11.0 21.0 37.5 54.0 70.5 86.5 63.0 Intensity after subtraction 0.0 6.5 16.5 33.0 49.5 66.0 82.0
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Plot curve (Use (Use Excel, new data) Calculate the conc using the linear equation 63 = 32.885x + 0.0404, gives x = 1.91ppm If lets say the original solution was diluted 1:2, then the actual concentration is multiplied by 2, ie 21.91 = 3.82ppm Solutions giving readings that exceed the maximum intensity must be diluted until it can be read in the range
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2. STANDARD ADDITIONS CALIBRATION METHOD Used when (a) difficult to duplicate the sample matrix or (b) for complex mixtures Objective: To minimize interferences and verify calibration curve validity Signals must be in linear region of working curve Solution without the analyte is assumed to have zero reading Approach: A series of standard (same as the analyte) of known quantity is added to a fixed amount of sample
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General Procedure
a) place known volume of unknown sample in multiple flasks b) Add increasing volume of known standard to each unknown sample

c) Fill the flasks to a constant known volume

d) Measure the analytical response for each sample e) Plot signals as a function of the added known analyte concentration The xx-intercept (y=0) yields [x]f which is used to calculate the unknown concentration from the equation:

Relevant equations etc

= =

Concentration of analyte in initial solution Concentration of analyte plus s tan dard in final solution signal from initial solution signal from final solution

[X ]i = [X ]f

V Vo

Standard addition equation:

[X ]i [S ] f + [ X ] f

IX IS+ X
V

V Total volume (V): [ X ] f = [ X ]i o V

V [S ] f = [S ]i S

V = Vo + VS , Vo = unknown initial volume, VS = added volume of s tan dard

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EXAMPLE 3 Determine the concentration of analyte in the sample using the following experimental data Concentration (ppm) X + 0.00 X + 10.00 X + 20.00 X + 50.00 X + 100.00 Response 5.7852 10.6227 15.4601 29.9723 54.1595 15 Plot the calibration curve Use the equation (y = mx + c) generated by excel to calculate the concentration The analyte in the sample obtained from graph is 12 ppm (Concentration = -x) Need to multiply by dilution factors (if any)
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3. INTERNAL STANDARD METHOD Objective: Objective: Correct for multiplicative errors - can improve accuracy and/or precision for nonnon-additive errors - will not correct for an additive error such as cloudiness of a sample solution in a UVUV-vis absorption experiment Approach: Approach: Add a known compound (element) in the same concentration to both samples and calibration standards. Plot the ratio of analyte signalsignal-to to-internal standard signal against concentration

A known amount reference (chemically and physically similar to that of the analyte) analyte ) is added into the standards and the sample Measure signal of analyte and IS The response signal is the ratio of the analyte signal to the reference signal Calibration curve: y-axis is the ratio of response x-axis is the analyte conc in the standards
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Requirement of ideal IS Sample has identical chemical and physical properties as analyte
unknown Analyte signal IS signal

Recording and Reporting Results

Distinction should be made between recording results and reporting results

Results should be recorded as they come (including results below detection limit and negative results) When reporting, the analyst should provide a statement of the method of editing negative values and results below the detection limit the uncertainty of measurement It is generally inappropriate to report negative results to customers! But unedited results should be retained if needed for statistical analysis
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Calibration Curve

concentration

Guidelines For Reporting Data

Analyte conc Report as

<3s 3s 3s - 10 s >10 s

Not detected Limit of detection Found but not quantitated Numerical value reported with associated uncertainty

Some terms associated with data/results Response Signal to noise ratio Limit of quantitation (LOQ) Limit of detection (LOD) Sensitivity Selectivity

Response
Response is the the way in which the signal (or result) of a method varies with the amount of material (analyte) being measured
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Recommended by The American Chemical Society, Committee on Environmental Improvement

SignalSignal -to to-Noise Ratio (S/N)

Noise: random variation in signal or

background Signal: net response recorded by a method for a sample Ratio of S/N 2 is considered to be the minimum needed for the reliable detection of a true signal from a sample
(Signal)

S/N is estimated by two methods: (1) Multiple determination of blank samples - Monitor background signal for a given period of time and determine variation from average by factor of 2 or 3 - Subtract the min. background signal from the max. background signal S/N = Signal/(Maxbackgrnd - Minbackgrnd)
(2) Estimation of bestbest-fit to calibration curves of the method
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(Noise)

Limits of Quantitation (LOQ)

LOQ - the lowest amount of analyte in a sample that can be quantitatively determined with precision & accuracy - Evaluated as, S/N = 10 or LOQ = 10s/S
where and s are std deviation and S is slope or sensitivity

Limits of Detection (LOD)

LOD - the lowest amount of analyte which can be detected but not necessarily quantitated Evaluated as, S/N = 3 or LOD = 3s/S
where and s are sd and S is slope or sensitivity
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Definition of LOD - the analyte conc giving a response of a confidence factor k higher than the std dev. of the blank LOD = ksb/m where m = calibration sensitivity k = 2 (for 92.1 % confidence level) or 3 (for 98.3% confidence level)
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Selectivity
Selectivity is the degree to which the method is free from interference by other species in the sample Selectivity coefficient (k) kB,A = mB/mA Species A Relative slopes of calibration curves indicate selectivity Species B S = mA(cA + kB,Acb) + Sbl
m is slope, bl is blank Eg. Interested in detecting A, but signal will be a combination of signal from presence of A and B

Sensitivity

ability to discriminate between small

differences in analyte concentration Reported as the change in signal per unit change in the amount of analyte
The SPME extraction method is more sensitive compared to the solvent extraction method

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Calibration sensitivity = slope of calibration curve, m Analytical sensitivity (g) = Slope/Std. dev = m/Ss
Slope and reproducibility of the calibration curve

VALIDATION OF ANALYTICAL METHODS

A process of performing several tests to verify that an analytical test is suitable for its intended purpose, purpose , and capable of providing valid data Analytical procedures are validated by a number of parameters

Method A

Method B

Accuracy Precision Sensitivity Detection limit Dynamic range

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Reproducibility linearity Ruggedness/ robustness

Linearity The range of concentrations of analyte for which the procedure gives results that are directly proportional to the concentration (amount) of analyte in the sample Ways of determining linearity - Use of Calibration or Standard Curve (Concentrations determination at the linear sections of the graph) - Triplicate (3) measurements at least - Regression, R2 > 0.998
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Accuracy Expression of the closeness of agreement between the accepted value (conventional true value or accepted reference value) and the value obtained by the method Accuracy can be determined in 3 ways - Recovery studies - use the procedure on a Standard Reference Material (SRM) and calculate % recovery (Method is a problem if % Recovery <90%) - Compare results using 2 or more independent methods (one accurate and validated) - Analyze spiked blank matrices with varying known amounts of a standard
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Precision The closeness of agreement between a series of measurements from multiple sampling Generally expressed as std. dev or RSD Repeatability: precision under the same operating conditions over a short time Intermediate Precision: precision within laboratories variations, under different days, different analysts, different equipment, etc Reproducibility: Precision between laboratories (eg. inter-lab collaborative studies, usually applied to standardization of methodology)
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Range The interval between the upper and lower concentrations of analyte in the sample for an analytical procedure that has a suitable level of precision, accuracy and linearity Limit Of Detection (LOD) LOD is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated (LOD = 3s/S) 3s/S)

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Limit Of Quantitation (LOQ) The lowest amount of analyte that can be quantitatively determined with suitable 10s/S) precision and accuracy (LOQ = 10s/S) - For assays of low levels of compounds in sample matrices (eg impurities and/or degradation products) Ruggedness or Robustness A measure of the methods capacity to remain unaffected by small, but deliberate variations in method parameters (Provides an indication of its reliability during normal usage)
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Specificity/Selectivity The ability to assess the analyte in the presence of components that are expected to be present (eg. matrix, impurities, etc) - It is not always possible to demonstrate that an analytical procedure is specific for a particular analyte (completely discriminate) - In this case a combination of two or more analytical procedures is recommended to achieve the necessary level of discrimination
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General Process of Method Validation

Determine precision, accuracy & detection limit when a single analyst uses the method to analyze a standard sample of known composition - Detection limit (determined by analyzing a reagent blank for each type of sample matrix for which a method will be used) - Precision (determined by analyzing replicate portions of a standard sample, various concentration with replicates) - Accuracy (evaluated by a tt-test) Blind Analysis - analytes concentration unknown to the analyst is analyzed several times and the mean determined (values should be within 3 std.dev, preferably 2 std.dev)
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EXTRA READINGS AND CALCULATIONS ON VALIDATION OF METHODS

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GOOD LABORATORY PRACTICE (GLP)

History

GLP - General laboratory operations that -

GLP was created in 1978 by the FDA

(US food and drug administration) - Why? In the early 70s FDA became aware of cases of poor laboratory practice all over the United States

need to be followed in any analysis It is a set of rules, operating procedures and practices established by an organization that is considered mandatory with view to ensure quality and correctness on results provided by a laboratory

In 1981 an organization called OECD

(organization for economic cocooperation and development ) produced GLP principles that are international standard
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Elements of GLP: - Standard Operation Procedures (SOP) - Quality Assurance (QA)

Standard Operations Procedure (SOP)

What is SOP and QA?

SOP - detailed written descriptions of

activities performed by a lab (eg sample handling and preparation, record keeping, etc)

Describes all steps taken during an analysis - Sample preparation, separation of interferents, standardization of the method, measurement, calibration

QA - a unit responsible for

implementing quality procedures and continuous assessment (including audits)
INSTRUMENT ANALYTICAL BALANCE GLASSWARES

CALIBRATION
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Good Measurement Practices (GMP)

Operations specific to a technique Provide instructions for maintaining, calibrating and using the equipment and instrumentation that form the basis for a specific technique eg GMP for titration describes how to calibrate a buret, how to fill a buret with titrant, correct way to read the volume of titrant in buret, and the correct way to dispense the titrant
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Time plot of a measured quantity that is

- eg. eg. Control Chart for analysis of serum Ca

Process Monitoring and Control Charts

assumed to be constant - Purpose: to ascertain measurements remains within acceptable range

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Reading the Control Chart Inner control limit: 2 Outer limit: 3 Inspect trends that lie in one direction Points on one side of the central line Indicates systematic error in measurement or control is in error Points outside the control limits Indicate presence of one or more determinate errors in measurement The analyst should check deterioration of reagents, instrument malfunction or other effects
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EXAMPLE: LIMIT OF DETECTION

Concentration of an analyte which gives an instrument signal (y) significantly different from the blank or background signal
LOD = 3 x S.D.Blank (abs/conc)
Slope

Procedure: Analyze a series of reagent blank being used (eg.7 replicates) and calculate its std. dev. Determine absorbance of known concentration of analyte (3 (3-5 times the estimated LOD) Example Problem: Series of blank solution was analyzed giving absorbance (A) values as tabulated in table. A standard solution of 1.5 ppm aspirin gave 0.095 A. A. Calculate the detection limit
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Answer: Limit Of Detection

Limit of Detection (LOD) = 3 SDBlank/slope = 3 x 0.002/(0.095/1.5) Conc. of 1.5 ppm = 0.095 mg/L gives 0.095 A *Slope = Absorbance/Concentration
Replicate 1 2 3 4 5 6 7 Mean SD Mean signal (blank) 0.005 0.006 0.005 0.010 0.004 0.006 0.008 0.006 0.002 Mean Signal 1.5 ppm 0.095 0.110 0.082 0.101 0.099 0085 0.095 0.095 0.010 47

EXAMPLE: PRECISION The precision of data is estimated by the deviation from the mean of the multiple analysis Replicate 5 ppm 1 2 3 4 5 6 7 SD 4.928 4.978 4.988 4.923 4.952 4.911 4.971 0.030 10 ppm 9.973 9.989 9.967 9.915 9.934 9.947 9.954 0.024 1 ppm (est DL) 0.993 0.972 0.984 0.961 0.952 0.963 0.974 0.021
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EXAMPLE: RECOVERY TEST (% Recovery) Accuracy is determined based on the recoveries of known amt of analyte spiked into the sample % recovery = (analyte conc in assay) assay) x 100 (analyte conc in sample) ) x 100 = (weight of analyte in assay assay) (weight of analyte from sample)
Repl 1 2 3 4 5 Mean Non spike (mg/L) 2.674 2.825 2.972 2.883 2.713 Spike (mg/L) 7.884 7.712 7.815 7.765 7.698 Standard spiked % Recovery 6 86.83% 6 87.38% 6 87.66% 6 80.71% 6 84.40% 85.29%
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EXAMPLE: REPEATABILITY - Done by performing replicates - Purpose to ensure that the method is working properly to reduce the s sampling error
(Method USEPA 3050 B)
Rep 1 2 3 Mean SD RSD [Sample](x %( w/w) 8.36 8.48 9.02 8.62 0.35 0.04 10-1) Rep 1 2 3 Mean SD RSD

(BS 11466)
[Sample] (x 10-1 ) %(w/w) 4.74 3.86 4.57 4.39 0.467 0.106

[Pb] in sediment sample was (8.62 0.04) x 10-1 % w/w)

[Pb] in sediment sample was (4.39 0.106) x 10-1)

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Good recoveries of analyte in the range 8080-90%

EXAMPLE: REPRODUCIBILITY

Different times
[Sample] %(w/w) 0.27 0.28 0.37 0.30 0.05 0.18 [Sample] %(w/w) 0.29 0.24 0.37 0.31 0.06 0.21

Identical samples

Rep 1 2 3 Mean SD RSD Rep 1 2 3 Mean SD RSD

EXAMPLE: = ([Std] x 0.0044 )/Abs of standard SENSITIVITY = (4.953 x 0.0044)/0.488 = 0.0405 mg/L Defined as the concentration of an element required to produce a signal of 1% absorption (0.0044 absorbance units) - can be determined by reading the absorbance produced by a known concentration of the element Replicate 1 2 3 4 5 6 7 Mean SD
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analyzed under different conditions (including times, equipment, analysts, or different laboratories) Reproducibility expresses the precision between laboratories (collaborative studies)
[Pb] in water sample was (0.31 (0.31 0.21)x10-1 mg/L

Concentration (mg/L) 4.935 4.984 4.925 4.951 4.982 4.923 4.973 4.953 0.027

Mean Signal (Abs) 0.484 0.495 0.483 0.492 0.498 0.491 0.479 0.488 0.007
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Example Method Limit of Detection

establish the lower end of the practical operating range of a method Estimation Det. Limit: 3g/L Optimum Conc. Range: 20-200g/L Method Detection Limit; MDL=3.14 X 0.0009 = 0.003 mg/L (student t-test at 99%)

Replicate

Conc (mg/L) 0.009 0.010 0.009 0.010 0.011 0.010 0.008 0.009 0.0009

Abs 0.015 0.020 0.017 0.021 0.025 0.018 0.012 0.018 0.0042

1 2 3 4 5 6 7 Mean SD

MDLMDL -The constituent concentration that went through complete method process

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