Analytical Chemistry:

Principles of advanced instrumental

analytical techniques

Lecturer: Tu Binh Minh

VNU Hanoi University of Science
Vietnam National University, Hanoi
 CONTENTS

1)Introduction to Analytical Chemistry

2)Classification of analytical methods
3)Principle of Calibration in Analytical chemistry
 LEARNING OUTCOMES

• To be able to identify the general principles and roles of analytical chemistry

• To comprehend and distinguish various quantification and calibration methods
in analytical chemistry
 Nomenclature – Thuật ngữ

English Tiếng Việt

1 Analytical chemistry, procedure, Hóa học phân tích, quy trình phân tích
process
2 Gravimetric/gravimetry method Phương pháp phân tích khối lượng/trọng lượng

3 Volumetric method Phương pháp phân tích thể tích

4 Calibration method Phương pháp chuẩn hóa, đường chuẩn

5 External, internal, standard addition phương pháp ngoại chuẩn, nội chuẩn, thêm chuẩn
calibration method
6 Sample preparation/treatment Xử lý mẫu

7 Sampling, sample collection Lấy mẫu, thu mẫu

8 Concentration Nồng độ, hàm lượng

9 Quality assurance/Quality control Bảo đảm chất lượng, kiểm soát chất lượng
 What is analytical chemistry?
• Analytical chemistry deals with separating, identifying, and quantifying the relative
amounts of the components of an analyte.
• Analyte = the thing to be analyzed; the component(s) of a sample that are to be
determined.

Qualitative analysis: Quantitave analysis:

what’s present? How much present?

Analytical chemistry
Main
subject

Characterisation analysis
What are chemical and physical
properties?
 Several different areas of analytical chemistry

1. Clinical analysis - blood, urine, feces, cellular fluids, etc., for use in diagnosis.

2. Pharmaceutical analysis - establish the physical properties, toxicity, metabolites, quality control, etc.

3. Environmental analysis - pollutants, soil and water analysis, pesticides.

4. Forensic analysis - analysis related to criminology; DNA finger printing, finger print detection; blood
analysis.

5. Industrial quality control - required by most companies to control product quality.

6. Bioanalytical chemistry and analysis - detection and/or analysis of biological components (i.e., proteins,
DNA, RNA, carbohydrates, metabolites, etc.).
Classification of Quantitative Methods of Analysis
Chemical methods
+ Gravimetric Method: based on the measurement of mass of analytes.
+ Volumetric Method: based on the measurement of volume of standards/analytes
* Low precision, low cost, long time, high concentration

Instrumental Method: use an instrumental technique to assay the amount of sample.

+ Electrochemical methods: based on redox reactions of analytes
+ Spectrophotometric methods: Based on interaction of samples and the light;
e.g. UV-VIS, AAS, mass spectrometry
+Separation methods: based on the techniques to separate components in
samples, while identify and quantify them; e.g. GC, HPLC, CE
* High precision, high cost, short time, low concentration, (ppm, ppb..)
 General steps in a chemical analysis & criteria for selection

• General steps: Criteria (Quality assurance/Quality

• 1. Select the analytical procedures control – QA/QC)
• 2. Sampling • Appropriate to analytical problems
• Limit of detection
• 3. Sample preparation • Precision
• 4. Analysis • Accuracy
• 5. Reporting and interpretation • Sensitivity
• Selectivity
• 6. Conclusions
• Ruggedness
Practical knowledge: Determination of caffeine in chocolate
 Sample preparation & Analysis
• Sample Preparation:
• Converting a representative sample into a suitable form for chemical
analysis
• Dissolve: in water, in solvents, in strong acids or alkaline
• Concentrate: increase concentration if too low
• Remove or mask interferes: to reduce the influence of other substances in sample
on the determination of analyte.
• Sample Analysis:
• Identify or measure the quantity of analyte in sample.
• Measure the signal of sample: several times of several identical portions
• Calibration curve: measure the signal of known samples
• Interpreting results with uncertainty
 Analytical results

Chromatogram of Chromatogram of Calibration curve

chocolate sample standard solution
containing 50
microgram/g of
theobromine and caffeine
Reporting and Conclusions
 Chemical concentrations

Molarity vs. molality

Percent composition (wt% ; vol %) w/w; v/v

Parts per Million and parts per Billion (ppm & ppb)
 Concentration
ppm = mg/L = µg/mL (for solution sample)
ppm = mg/kg = µg/g (for solid sample)
Example: 1ppm Cu solution ↔ 1mg/L Cu solution ↔ In 1L of solution contains 1 mg Cu.
ppb = µg/L = ng/mL
= ng/g = µg/kg
Example: The concentration of dioxin in soil at the vicinity of Bien Hoa airbase is 20 ppb ↔
20 µg/kg ↔In 1 kg soil contains 20 µg of dioxin
 Preparing solutions
• Prepare from pure solid or liquid
• Weigh out the correct mass of reagent and dissolve in a volumetric flask
• Dilution
• Prepare dilute solutions from concentrated solutions.
• Dilution formula
Example
A) A laboratory procedure calls for 250
mL of an approximately 0.10 M solution
of H2C2O4. Describe how you would
prepare this solution using solid
H2C2O4•2H2O.

B) A laboratory procedure calls for 250

mL of an approximately 0.10 M solution
of HCl. Describe how you would
prepare this solution using a stock
solution of concentrated HCl (C% =
37.5%; d = 1.19 g/ml).
 Gravimetric Analysis: Stoichiometry calculations

• Gravimetry: chemical analysis bases on the weighing a final product

Tablet containing FeC4H2O4

Dissolve by HCl Fe2+

Oxidize to Fe3+ by H2O2 Fe3+

Precipitate with NH4OH

Fe(OH)3
Filter the precipitate

Heat in furnace Fe2O3 m(g)

If m = 0.277 g à what is the
Weigh average mass of iron per tablet?
 Volumetric analysis: Titration

• Volumetric analysis: measure the volume of reagent needed to

react with analyte
• Reagent: titrant
• Equivalence point: stoichiometric reaction
• End point: when we stop titration
• Detect the end point:
• Using indicator that changes color
• Detect a change in voltage or current
• Monitor absorption of light
• Titration error: difference between the
end point and equivalence point
Example Titration
 Calibration in Analytical chemistry

1) Calibration curves and calibration methods

1) - Constructing a calibration curve
2) - Linear regression to find the best fit straight line: Method of least square
3) - Calibration methods: External method, standard addition, Internal
standard
2) Quality assurance
3) Method validation
 Calibration curves

• Calibration curve: response of analytical method to

known quantities of analyte.
- Standard solutions: contain known
concentration of analytes
- Blank solution: contain all reagents and
solvent in analysis but no analytes
 Constructing a calibration curve

• Step 1: Prepare known samples of analyte covering a range of concentrations expected

for unknowns. Measure the response of the analytical procedure to these standards to
generate data
• Step 2: Subtract the average absorbance of the blank samples from each measured
absorbance to obtain corrected absorbance.
• Step 3: Make a graph of corrected absorbance versus quantity of protein analyzed. Use
the least-squares procedure to find the best straight line through the linear portion of
the data.
Linear equation of calibration curve
A = 0.01630 xC + 0.0047

Linear range: concentration range has linear response

Dynamic range: concentration range has measured response
 Methods of calibration
• External standard: prepare standards separate from samples.
Disadvantage: matrix of standard can be different from sample à may introduce determinate error.
• Standard addition:
- Add (spike) standard to the sample.
- Single standard addition
- Multiple standard addition (successive addition)
Advantage: minimize effect of matrix
Disadvantage: used for only one sample one time.
• Internal standard:
- Used when instrument response varies from run to run, sample loss during sample preparation
prior to analysis.
- Add another species different than the analyte to all samples and standard.
- Instead of using Sa use the ratio of Sa/Sis
 Single standard addition

• Sample: concentration [X]I, signal Ix

• Add unknown amount of standard in sample: Sf, Is+X
• Since signal is proportional to concentration
Successive standard addition to one solution

Add successive small volumes of

standards in one solution
 Analytical method: Spectroscopy & spectrophotometry

Spectroscopy is an analytical technique which helps determine structure.

It destroys little or no sample. The amount of light absorbed by the sample is measured as wavelength is varied.

• Types of spectroscopy:
• Infrared (IR) spectroscopy measures the bond vibration frequencies in a molecule and is
used to determine the functional group.
• Mass spectrometry (MS) fragments the molecule and measures the masses.
• Nuclear magnetic resonance (NMR) spectroscopy detects signals from hydrogen atoms
and can be used to distinguish isomers.
• Ultraviolet (UV) spectroscopy uses electron transitions to determine bonding patterns.
The Spectrum and Molecular Effects
 Spectrophotometry

Spectroscopy and Spectrophotometry

§ Spectroscopy general study of interaction of matter with
electromagnetic waves.
§ Spectrophotometry is the quantitative measurement of light spectra
reflection and transmission properties of materials as function of the
wavelength. Spectrophotometry refers to the quantitative
measurement.
 Electromagnetic radiation
• Electromagnetic radiation (light): form of energy,
described by the properties of both waves and
particles.
energy
propagation

Wavelength l (m)

Frequency h: Planck’s constant =

6.626 x 10–34 J. s.
Wave number
(cm-1)
Interaction of Radiation and Matter

- Absorption
- Luminescence
Intera
- Emission
 Molecular UV-VIS spectroscopy: Transmittance, Absorbance and Beer
law

Irrandiance (P) energy per unit time per unit area in the light beam
(W/m2), also called intensity or radiant power

Due to absorption of sample: P < P0

 Beer law
Absorbance is proportional to concentration C of the light-
absorbing species in sample

A: absorbance
b: path length in cm
C: concentration in mol/L (M)
e: molar absorptivity (cm-1 M-1)

e: characteristics for each substance, dependent on wavelength

How to measure absorbance

-Irradiate light through cuvette containing sample.

-Choose suitable wavelength
- Measure P0: using cuvette with blank reagent
- Measure P: using cuvette with sample solution
- Determine A
Sample holder in AAS: cuvette

- UV-Vis light: glass, plastic, quartz cuvette

- Infrared light: KBr or NaCl cuvette

- Measuring gas: use long cuvette (10cm)
 Example

1. A sample solution stored in curvette 1 cm having transmittance T of 80% at certain

wavelength. Given the molar absorptivity at this wavelength is 2,0 L cm-1 mol -1 .
How much the concentration of sample in the solution.

Solution:
The transmittance is 80% à T = 0,8.
According to Beer Law: A = ε.b.C = - log T
Replace values in the Beer Law equation:
C = 0,10/2,0 = 0,048 M/l
Example
A solution containing 1.0 mg ion SCN- in 100 ml having a transmittance T of 70% at a given
wavelength.
-How much absorption A of this solution at this wavelength?
-Calculate T value of the concentration of the solution increase 4 times.

Transmittance T = 0,70
a) A = - log T = - log (0,70) = 0,155
b) Apply Beer Law:
0,155 = εb (0,010 g/l)
à εb = 15,5 l/g
If concentration increases 4 times:
A = 15,5 l/g (4 x 0,010g/l) = 0,620
Log 1/T = 0,620 Þ T = 0,240
Practical knowledge: procedure for determination of Fe in
blood (serum) sample
 Practice problem: determination of Phosphorus P in human urine
One common way to determine phosphorus in urine
is to treat the sample after removing the protein with
molybdenum (VI) and then reducing the resulting
12-molybdophosphate complex with ascorbic acid
to give an intense blue-colored species called molybdenum
blue. The absorbance of molybdenum blue
can be measured at 650 nm. A 24-hour urine sample
was collected, and the patient produced 1122 mL
in 24 hours. A 1.00 mL aliquot of the sample was
treated with Mo(VI) and ascorbic acid and diluted
a. Find the slope, intercept, and Construct a calibration
to a volume of 50.00 mL. A calibration curve was
curve.
prepared by treating 1.00 mL aliquots of phosphate
b. Determine the concentration of phosphorus in ppm in the
standard solutions in the same manner as the urine
urine sample
sample. The absorbances of the standards and
c. What mass in grams of phosphorus was eliminated
the urine sample were obtained at 650 nm and the
per day by the patient?
following results obtained:
 Practical problem: determination of Ammonia: Ammonia can be determined
spectrophotometrically by reaction with phenol in the presence of hypochlorite (OCl-)

A 4.37-mg sample of protein was chemically digested to convert its

nitrogen into ammonia and then diluted to 100.0 mL. Then 10.0 mL
of the solution were placed in a 50-mL volumetric flask and treated
with 5 mL of phenol solution and 2 mL of sodium hypochlorite
solution. The sample was diluted to 50.0 mL, and the absorbance at
625 nm was measured in a 1.00-cm cuvet after 30 min. For reference,
a standard solution was prepared from 0.0100 g of NH4Cl
(FM 53.49) dissolved in 1.00 L of water. Then 10.0 mL of this standard
were placed in a 50-mL volumetric flask and analyzed in the same
manner as the unknown. A reagent blank was prepared by
using distilled water in place of unknown (a) Calculate the molar absorptivity (ε) of the blue product.
(b) Calculate the weight percent of nitrogen in the protein
 Analysis of mixture

Absorbance has additive property