Instrumental Analysis – Calibration Methods

  • Instrumental Analysis

  • The course is designed to introduce the student to modern methods of instrumental analysis

  • In modern analytical chemistry. The focus of the course is in trace analysis, and therefore methods for the identification, separation and quantitation of trace substances will be described.

  • Scope and Relevancy of

    Instrumental Analysis

  • Instrumental Methods

  • Instruments

  • Calibration Methods

  • Method Validation

  • Specificity

  • Linearity

  • Accuracy

  • Precision

  • Range

  • Limits of Detection and Quantitation

  • Method Validation – Specificity

  • How well an analytical method distinguishes the analyte from everything else in the sample.

  • Baseline separation

  • Method Validation- Linearity

  • How well a calibration curve follows a straight line.

  • R2 (Square of the correlation coefficient)

  • Method Validation- Linearity

  • Method Validation- LOD and LOQ

  • Limit of Detection (LOD)

  • Limit of Linear Response (LOL)

  • Useful Range of an Analytical Method

  • Method Validation- Linearity

  • Method Validation- Accuracy and Precision

Precision – reproducibility

  • Method Validation- LOD and LOQ

  • Standard Addition

  • Standard addition is a method to determine the amount of analyte in an unknown.

–In standard addition, known quantities of analyte are added to an unknown.

–We determine the analyte concentration from the increase in signal.

  • Standard addition is often used when the sample is unknown or complex and when species other than the analyte affect the signal.

–The matrix is everything in the sample other than the analyte and its affect on the response is called the matrix effect

  • The Matrix Effect

  • The matrix effect problem occurs when the unknown sample contains many impurities.

  • If impurities present in the unknown interact with the analyte to change the instrumental response or themselves produce an instrumental response, then a calibration curve based on pure analyte samples will give an incorrect determination

  • Calibration Curve for Perchlorate with Different Matrices

  • Calculation of Standard Addition

  • The formula for a standard addition is:

 [X] is the concentration of analyte in the initial (i) and final (f) solutions, [S] is the concentration of standard in the final solution, and I is the response of the detector to each solution.

  • But,

 If we express the diluted concentration of analyte in terms of the original concentration, we can solve the problem because we know everything else.

  • Standard Addition Example

  • Serum containing Na+ gave a signal of 4.27 mv in an atomic emission analysis. 5.00 mL of 2.08 M NaCl were added to 95.0 mL of serum. The spiked serum gave a signal of 7.98 mV.  How much Na+ was in the original sample?

  • Standard Additions Graphically

  • Internal Standards

  • An internal standard is a known amount of a compound, different from the analyte, added to the unknown sample.

  • Internal standards are used when the detector response varies slightly from run to run because of hard to control parameters.

e.g. Flow rate in a chromatograph

  • But even if absolute response varies, as long as the relative response of analyte and standard is the same, we can find the analyte concentration.

  • Response Factors

  • Internal Standard Example

  • In an experiment, a solution containing 0.0837 M Na+ and 0.0666 M K+ gave chromatographic peaks of 423 and 347 (arbitrary units) respectively. To analyze the unknown, 10.0 mL of 0.146 M K+ were added to 10.0 mL of unknown, and diluted to 25.0 mL with a volumetric flask. The peaks measured 553 and 582 units respectively.  What is [Na+] in the unknown?

  • First find the response factor, F

  • Internal Standard Example (Cont.)

  • Now, what is the concentration of K+ in the mixture of unknown and standard?

  • Now, you know the response factor, F, and you know how much standard, K+ is in the mixture, so we can find the concentration of Na+ in the mixture.

  • Na+ unknown was diluted in the mixture by K+, so the Na+ concentration in the unknown was:

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