5.0 Quality Assurance
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1 5.0 Dr. Fred Omega Garces Analytcal Chemstry 25 Natural Scence, Mramar College
2 Bascs of s what we do to get the rght answer for our purpose QA s planned and refers to planned and systematc producton processes that provde confdence n a product's sutablty for ts ntended purpose. It s mportant to realze also that qualty s determned by the ntended users, clents or customers, not by socety n general: t s not the same as 'epensve' or 'hgh qualty. In a laboratory, raw data, treated data and results are the products. What are the procedures to nsure that these are relable nformaton wthout spendng etra money or tme to obtan a more accurate or more precse answer than necessary. 2 Use Objectve- Analyss meet customer s needs. Specfcaton- How good results need to be and what precautons to be aware. Assessment Collect data, crunch and verfy that results meet objectve.
3 Use Objectves Does the result meet the end user s need? Use objectve to answer the questons: Why do you want the data and results? How wll the results be used? By wrtng clear and concse objectve, the data and results wll be prevented from beng used napproprately. In short, The objectve should state the purpose for whch results are to be used. 3
4 Specfcatons Informaton about how good numbers need to be and what precautons are requred n the analytcal procedure. Eample of nformaton mght nclude- -Samplng requrements -Accuracy and precson -Rate of false results (False postve or false negatve) -Selectvty (Specfcty, Standard Reference Materal) -Senstvty (Detecton lmts) -Acceptable blank values (method blank, reagent blank, feld blank) -Recovery of fortfcaton (spke recovery, matr) -Calbraton checks -Performance Test Samples (Qualty control samples. Blnd samples). -Standard Operatng procedures (Chan of custody, nst. mantenance) 4
5 Spke Recovery Sgnal of analyte ncrease or decrease because of unknown substance. Matr effect (everythng other than analyte) can cause nterference. Spke recovery s used to correct for matr effect. Recovery [(C spked C unspked ) / C added ] : Arsenc n the form of AsO 3-3 and AsO 3-4 can contamnate drnkng water. Pure water (no arsenc) s spked wth 0.40 µg arsenate/l. Seven replcate determnaton gave 0.39, 0.40, 0.38, 0.4, 0.36, 0.35 and 0.39 µg/l. What s the percent recovery and the concentraton detecton lmt (µg/l). Average Arsenc found: y blank 0, std dev Recovery C - C spked unspked C added 0.40 Detecton Lmt y dl y blank + 3s std dev (s) 0 + 3(0.02) µg/l 5
6 Assessment Ths s the process of () collectng data to show the analytcal procedures are operatng wthn specfed lmts and (2) verfy that the fnal results meet the use objectve. Key to assessment s documentaton.e., lab notebook. Standard protocols provde the drectons on what must be documented and the format n whch t must be done. Control charts can be used to montor performance on blanks, calbraton checks and spked samples. Assessment answers the queston as to whether the specfcaton was acheved. Ths s carred out by comparng data and results wth specfcaton. By valdatng the documentaton of procedures and results, and then addressng the objectve agan and determnng f t was met. 6
7 Process Questons Use objectves -Why do you want the data and results and how wll you use the results? Actons -Wrte use objectve 7 Specfcaton -How good do the numbers have to be? Assessment -Were the specfcatons acheved -Wrte specfcatons -Pck method to meet specfcatons -Consder samplng, precson, accuracy, selectvty, senstvty, detecton lmts, robustness, rates of false results -Employ blanks, fortfcaton, calbraton checks, qualty control samples and control charts to montor performance. -Wrte and follow standard operatng procedures -Compare data and results wth specfcatons. -Document procedure and keep records sutable to meet use objectve. -erfy the use objectves were met.
8 aldaton of Procedure (Method aldaton) If a new analytcal method s used or an old method s used for new type of sample, then the procedure must be valdated. In the botech ndustry, method valdaton requres- -selectvty: dstngush analyte from everythng else. -accuracy: nearness to the truth. Check wth certfed reference materal. -precson: how well replcated measurements agree (standard devaton) -lnearty (Calbraton curve): Test wth standard or correlaton factor R 2 -range: concentraton nterval for relablty -robustness: analytcal method unaffected by change n operatng parameters. -lmts of detecton (lower lmts of detecton; mn detectable conc. (3s) / m, s std dev, m slope) -lmt of quanttaton. (lower lmt quanttaton 0 s / m) smallest amt wth accuracy 8
9 Range: Lnear Dynamc The concentraton nterval over whch lnearty, accuracy and precson are acceptable. Range: Concentraton range over whch lnearty, accuracy and precson meet specfcaton. Lnear range: Concentraton range over whch calbraton curve s lnear. Dynamc range: Concentraton range over whch there s measureable response. Lnearty: Correlaton Coeffcent, R 2 > R 2 < 0.98 (mpurty) Accuracy: Spke recovery (mpurty) Precson, Interlaboratory: (mpurty) 9
10 Word about False Postve or False Negatve Detecton Lmts and Quanttaton. Gaussan profle for blank and the sample shown. Ths s the profle showng a detecton lmt wth 99 chance of beng better than the blank wth no sample or that there s only a chance that the blank s above the detecton lmt. Ths may be nterpreted that there s only a chance of a false postve. 0
11 Detecton Lmt The lower lmt of detecton s the smallest quantty of analyte that s sgnfcantly dfferent from blank. Procedure that produces a detecton lmt wth ~99 chance of beng greater than blank. Prepares sample that are -5 tmes the detecton lmt 2. Measure sgnal mnmum of > 7 tmes. Then calc. std devaton 3. Measure blanks. The mn detectable sgnals s as follow Sgnal detecton lmt: y dl y blank + 3s Corrected sgnal: y sample - y blank, s proportonal to sample concentraton Calbraton lne: y sample -y blank m sample concentraton Where, y sample sgnal observed and m slope mnnmum lmt: Mnmum detecton conc. 3s m (stll too small for accuracy) 3 > nose Lower lmt of quanttaton Quanttaton lmt. 0s m 0 > nose
12 Standard Addton Standard addton s approprate when calbraton curves cannot be generated. Ths may be due to the complety or unknown nature of the sample matr. A known quantty of analyte s added to the sample and an ncrease n the sgnal s measured. The relatve ncrease provdes nformaton on the amount of analyte n the orgnal sample. Std Addton Eqn: Conc Analyte (unknown) Conc Analyte (+ std n mure) Sgnal (unknown) Sgnal (mture) [X] [X] f + [S] f I X I S+X Where, s the ntal concentraton whch gves a sgnal, and [X] f s the fnal concentraton of the unknown after addng the standard. The known concentraton of the standard s [S] f and the mture gves the sgnal I s+. The dluton factor s accounted for by the followng equatons- [X] f o f Dluton factor [S] f [S] so f Dluton factor 2
13 Graphcal Procedure: Standard Addton Eample: tamn-c measured by Electrochem n 50.0mL sample of juce. Sample gave sgnal of 2.02 µa. A standard addton of.00ml of 29.4 mm t gave sgnal of 3.79µA. What s the concentraton of tamn C [X] f 5.0, [S] f.0 [S] [S] [X] 0 + [S] f s 0 f [S] s 0 f - 0 f [S] s 0 f - 0 f [29.4mM] mm 3
14 Graphcal Procedure: Standard Addton Eample: tamn-c measured by Electrochem n 50.0mL sample of juce. Sample gave sgnal of 2.02 µa. A standard addton of.00ml of 29.4 mm t gave sgnal of 3.79µA. What s the concentraton of tamn C [S] [X] f 5.0, [S] f.0 [S] 5.0 [X] 0 + [S] f s 0 f [S] s 0 f - 0 f [S] s 0 f - 0 f [29.4mM] mm 4
15 Graphcal Procedure: Standard Addton The graphcal procedure to standard addton s much more accurate. Ths procedure nvolves makng a seres of standard addton that ncreases the sgnal of the orgnal sample by a factor of.5-3. Y b + m Frst Dervaton [X] f +[S] f ([S] f +[X] f ), [S] [S] f s, [X] [X] f 0 I [S] s [X] + 0 I ( 0 * [X] * [S] ) s , Substtute for [S] f and [X] f Factor out o / I +s o Y I ( + *[S] s o [X] + [X] - * - ) o, I + b ( I + *[S] s - [X] * - ) o, m ( I * [S] * o Y 0, s o , Multply bot sde by / o Dstrbute rght sde by / Y m + b Set (/ o ) 0 Solve for, - [S] ( s / o ) whch s 5
16 Graphcal Procedure: Standard Addton The graphcal procedure to standard addton s much more accurate. Ths procedure nvolves makng a seres of standard addton that ncreases the sgnal of the orgnal sample by a factor of.5-3. Y b + m Second Dervaton [X] f +[S] f [X] o ' ' +[S] s ' ' I ( ( * [S] * s [X] * * ) ) , ( * * ) ,, ( + * - * ) - I ( [S] * s o, [X] * ) , ( * * ) 0 + ( + -'* - -'*,) - o, Multply both sde by / o ( + * - * ) - I ( [S] * s o, [X] * ) + ( + -* - -* - +,), o ( * * ) 0 + ( + -* -' -*,) -' o, ( + * - * ) - I ( + [S] * s - o, [X] * ) - + o, ' ' Multply through and cancel I ( + +s * - * ) - o, Y I ( + [S] * s - ' [X] * ) - ' o, m + I Y 0, Y m + b Set (/ o ) 0 Solve for, -[S] ( s / o ) whch s 6
17 Graphcal Procedure: Standard Addton The graphcal procedure to standard addton s much more accurate. Ths procedure nvolves makng a seres of standard addton that ncreases the sgnal of the orgnal sample by a factor of.5-3. I s + f o + I [S] s [X] o y 0 [X] [S] s ( whch s ntercept o ' functon to plot on y-as functon to plot on -as Left part of equaton: I s+ s the sgnal measured for sample contanng unknown plus standard. f / o s the fnal volume dvded by the ntal volume of the sample. I s+ ( f /o) s the corrected sgnal or the sgnal that would have been measured wthout the dluton Rght part of equaton: [S] s the concentraton of the standard pror to addng t to the sample s s the volume of standard added o s the ntal volume of the analyte unknown before addng standard A graph of I s+ (/o) on the y-as versus [S] ( s / o ) on the -as gves an ntercept of the ntal concentraton for unknown 7
18 Standard Addton: Eample Equal volumes of unknown are ppetted nto several volumetrc flasks. Increasng volumes of standard are added to each flask and each dluted to 50mL. o 50 ml [S] 279 mm I s+ o functon to plot on y-as I + [S] s o I functon to plot on -as Std Devaton of -ntercept s y2 σ y + n m2 ( )2 m 8
19 Internal Standards The technque of nternal standard uses a known amount of a compound, dfferent from analyte, that s added to an unknown. The sgnal from the analyte s compared wth the sgnal from the nternal standard to determne the amount of analyte. An nternal standard n analytcal chemstry s a chemcal substance that s added n a constant amount to samples, the blank and calbraton standards n a chemcal analyss. Ths substance can then be used for calbraton by plottng the rato of the analyte sgnal to the nternal standard sgnal as a functon of the analyte concentraton of the standards. Ths s done to correct for the loss of analyte durng sample preparaton or sample nlet. The nternal standard s a compound that matches as closely, but not completely, the chemcal speces of nterest n the samples, as the effects of sample preparaton should, relatve to the amount of each speces, be the same for the sgnal from the nternal standard as for the sgnal(s) from the speces of nterest n the deal case. Addng known quanttes of analyte(s) of nterest s a dstnct technque called standard addton, whch s performed to correct for matr effects. (Wkpeda) Area of Analyte sgnal Concentraton of analyte F area of standard sgnal concentraton of standard 9 A [X] F A s [S]
20 Internal Standards: Eample A soluton contanng 3.47 mm X (analyte) and.72 mm S (standard) gave peak areas of and respectvely, n a chromatography analyss. Then.00mL of 8.47 mm S was added to 5.00mL of unknown X, and the mture was dluted to 0.0 ml. Ths soluton gave peak areas of and 4.43 for X and S, respectvely. ) Calculate the response factor for the analyte ) Fnd the concentraton of S (mm) n the 0.0mL of med soluton ) Fnd the concentraton of X (mm) n the 0.0 ml of med soluton v) Fnd the concentraton of X n the orgnal unknown. A 3.473, [X] 3.47, A s 0.222, [S].72 A [X] F A s [S] A ) [X] F A s [S] [3.47 mm] F [.72 mm] F ml ) [S] 8.47mM mm 0.0 ml ) A [X] F A s [S] [X] [X] 6.6 mm 0.847mM 20 v) the orgnal concentraton of [X] was twce as great at the dluted concentraton, so [X] 2.3 mm
21 Internal Standards: Eample A soluton contanng 3.47 mm X (analyte) and.72 mm S (standard) gave peak areas of and respectvely, n a chromatography analyss. Then.00mL of 8.47 mm S was added to 5.00mL of unknown X, and the mture was dluted to 0.0 ml. Ths soluton gave peak areas of and 4.43 for X and S, respectvely. ) Calculate the response factor for the analyte ) Fnd the concentraton of S (mm) n the 0.0mL of med soluton ) Fnd the concentraton of X (mm) n the 0.0 ml of med soluton v) Fnd the concentraton of X n the orgnal unknown. A 3.473, [X] 3.47, A s 0.222, [S].72 A [X] F A s [S] A ) [X] F A s [S] [3.47 mm] F [.72 mm] F ml ) [S] 8.47mM mm 0.0 ml ) A [X] F A s [S] [X] [X] 6.6 mm 0.847mM 2 v) the orgnal concentraton of [X] was twce as great at the dluted concentraton, so [X] 2.3 mm
22 Summary Qualty assurance s what we do to get the rght answer for our purpose. The process ncludes: () Use objectve, (2) Specfcaton whch may nclude requrement for samplng, accuracy, precson, specfcty, detecton lmt, standards and blank values. Meanngful analyss must frst collect representatve sample and these samples can be analyze by standard addton or addton of standards. Method valdaton s the process of provng that an analytcal method s acceptable for ts ntended purpose. 22
23 Answer to Eercse P5: Average Arsenc found: y blank 0, std dev P6 A 3.473, [X] 3.47, A s 0.222, [S].72 Recovery C - C spked unspked C added 0.40 Detecton Lmt y dl y blank + 3s std dev (s) 0 + 3(0.02) µg/l 50.0 [X] f 5.0, [S].0 [S] f [S] ) A [X] F A s [S] ) [S] 8.47mM ) A [X] F A s [S] [3.47 mm] F [.72 mm] F ml 0.0 ml [X] mm [X] 6.6 mm 0.847mM v) the orgnal concentraton of [X] was twce as great at the dluted concentraton, so [X] 2.3 mm [X] 0 + [S] f s 0 f [S] s 0 f I +s - 0 f [S] s 0 f I +s - 0 f [29.4mM] mm 23
y and the total sum of
Lnear regresson Testng for non-lnearty In analytcal chemstry, lnear regresson s commonly used n the constructon of calbraton functons requred for analytcal technques such as gas chromatography, atomc absorpton
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