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:

Pulsed Nuclear Magnetic Resonance Solid Fat Content ( Dr. A. Kaya )




The crystallinity of a fat at given temperature and time is characterised with the solid fat content (SFC). Atomic nuclei are spinning, resulting in a magnetic momentum. Brought in a magnetic field, the nuclei show precession with the so-called Lamor frequency around the lines of magnetic flux. During a NMR measurement, an electromagnetic pulse and such energy is sent through the sample. The spins get deflected. The magnetic momentum of all atomic nuclei are summed, inducing a signal in the receiving inductor. As soon as the energy drops the spins will relax to their original orientation. Two different spin relaxation behaviours can be investigated, the so-called T1- and T2- relaxations. T1-relaxation gives the time needed for the spins to come back to their original orientation. The T2-relaxation, also called spin-spin-relaxation, gives the decay of the the accumulated signal. The T1- and T2- relaxations are dependent on different material and the state of aggregation. The determination of the SFC for fats is possible using two different methods: the direct and the indirect method. Only the direct method is described here: For determining the SFC for fats by direct method, the signal of the solid and the liquid phase is measured and compared to each other. A calibration of the NMR apparatus is needed. K-, F- and O-values are calculated, where the K-value describes the slope of the solid signal domain. The F-value is used for the extrapolation of the signal to the y-axis, as the signal is not counted until a certain dead time. As the F-factor is dependent on the crystal polymorph and on the temperature, a systematic error occurs using the same calibration. For the value of the solid phase the signal measured after 11 μs, and for the value of the liquid phase the signal measured after 70 μs, is used and averaged from several scans.

Uv-Vis Spectrophotometry ( Dr. Sibel FADILOĞU )


Instructor: Dr. Sibel Fadıloğlu

Spectrophotometry is an important branch of spectroscopy that focuses on the technique of measurement. In this technique, the amount of light that a sample absorbs at a particular wavelength is measured and used to determine the concentration of the sample by comparison with appropriate standards or reference data.

Ultraviolet and visible spectrometers have been in general use for the last 35 years and over this period have become the most important analytical instrument in the modern day laboratory. In many applications other techniques could be employed but none rival UV-Visible spectrometry for its simplicity, versatility, speed, accuracy and cost-effectiveness.

The basic parts of a spectrophotometer are a light source, a holder for the sample, a diffraction grating or monochromator to separate the different wavelengths of light, and a detector. The radiation source is often a Tungsten filament (300-2500 nm), a deuterium arc lamp, which is continuous over the ultraviolet region (190-400 nm) or more recently, light emitting diodes (LED) and Xenon arc lamps for the visible wavelengths.

A spectrophotometer can be either single beam or doble beam. In a single beam instrument, all of the light passes through the sample cell. P 0 must be measured by removing the sample This was the earliest design, but is still in common use in both teaching and industrial labs. In a double-beam instrument, the light is split into two beams before it reaches the sample. One beam is used as the reference; the other beam passes through the sample. The reference beam intensity is taken as 100%

Transmission (or 0 Absorbance), and the measurement displayed is the ratio of the two beam intensities. Some double-beam instruments have two detectors (photodiodes), and the sample and reference beam are measured at the same time. In other instruments, the two beams pass through a beam chopper, which blocks one beam at a time. The detector alternates between measuring the sample beam and the reference beam in synchronism with the chopper.

Samples are typically placed in a transparent cell, known as a cuvette. Cuvettes are typically rectangular in shape. The most widely applicable cuvettes are made of high quality fused silica or quartz glass because these are transparent throughout the UV, visible and near infrared regions. Glass and plastic cuvettes are also common, although glass and most plastics absorb in the UV, which limits their usefulness to visible wavelengths. For convenience of reference, definitions of the various spectral regions have been set by the Joint Committee on Nomenclature in Applied Spectroscopy (Table 1).