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1.

CALCULATION – lab
a. Standard solution
m1v1 = m2v2
m1 - concentration of stock solution
v1 - volume of stock solution
m2 - final concentration of new solution
v2 - final volume of new solution

for concentration 0.01 mg/L


m1 - 1000 mg/L
v1 - ?
m2 - 0.01 mg/L
v2 - 0.1 L

m1v1 = m2v2
(1000) (V1) = (0.01) (0.1)
V1 = 1 µL

Working solution needed due to concentrated stock solution


Assume M2 = 200 mg/L

m1v1 = m2v2
(1000) (V1) = (200) (0.1)
V1 = 0.02 L

Standard solution
m1v1 = m2v2
(200) (V1) = (0.01) (0.1)
V1 = 5 µL

b. Dilution factor
𝑓𝑖𝑛𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒
Dilution factor =
𝑠𝑜𝑙𝑢𝑡𝑒 𝑣𝑜𝑙𝑢𝑚𝑒

𝑓𝑖𝑛𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒
Solute volume =
𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟

= 30 mL

c. Concentration in elements
i. River water
𝑓𝑖𝑛𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒
Dilution factor =
𝑠𝑜𝑙𝑢𝑡𝑒 𝑣𝑜𝑙𝑢𝑚𝑒

100𝑚𝐿
=
50𝑚𝐿

=2

Concentration in sample = concentration in sample solution x DF


= 0.4 ppm x 2
= 0.8 ppm
ii. Fish sample
𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑖𝑛 𝑠𝑎𝑚𝑝𝑙𝑒 𝑥 𝑓𝑖𝑛𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒
Concentration in sample =
𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡
(1.3 𝑝𝑝𝑚)(0.05 𝐿)
= 5000 𝑚𝑔
= 1.3 x 10-3 ppm

2. DISADVANTAGES OF DRY ASHING METHOD – test


Dry Ashing is usually performed by placing the sample in an open inert vessel and destroying the
combustible (organic) portion of the sample by thermal decomposition using a muffle furnace

ADVANTAGES DISADVANTAGES
1. Losses due to retention to the ashing
1. The ability to decompose large sample sizes.
container.
2. The need for little or no reagents.
2. Losses due to volatilization.
3. The technique is relatively safe.
3. Contamination from the ashing
4. The ability to prepare samples containing
container.
volatile combustion elements such as sulfur,
4. Contamination from the muffle furnace.
fluorine and chlorine (the Schöniger oxygen
5. Physical loss of 'low density' ashes when
flask combustion technique is very popular in
the muffle door is opened (air currents).
this case).
6. Difficulty in dissolving certain metal
5. The technique lends itself to mass production
oxides.

3. ADVANTAGES OF WET DIGESTION

ADVANTAGES DISADVANTAGES
1. Almost no losses of analytes
2. Minimal contamination 1. Some materials are particularly resistant
3. Much safer than decomposition using HClO4 to acid digestion, e.g., certain rocks,
4. No difficulties associated with the use of H2SO4 mineral oxides, phosphates, and some
5. Low consumption of acids iron alloys
6. Fast decomposition (microwaves) – automation 2. Introduction of additional salts into the
7. Quicker method final solution
8. No difficult dissolution of ashed materials

4. ADVANTAGES OF ICP OVER AAS


The basic difference between the two techniques is that one relies upon an atomic absorption
process while the other is an atomic/ionic emission spectroscopic technique. The next essential
difference is the means by which the atomic or ionic species is generated. A combustion flame or
graphite furnace is typically used for AA while ICP-ES uses a plasma
Induced Coupled Plasma is better system to use to determine metal element in a sample because it
offers:
 high productivity
 very low detection limits
 high stability
 measurement of many elements in one run
 measurement of one element in wide range – it means that you can measure for example lead in
low concentration in one sample and then in high concentration for next sample
But, the ICP-MS instrument is perhaps 4 to 5 times more expensive than Flame AAS. It uses a lot of
Argon (about 15 L/min). It requires ultra-pure reagents and the maintenance and operation of ICP-MS is
far more complicated than AAS
5. WHY METHOD VALIDATION IS IMPORTANT?
 To prove what we claim is true
 To minimize analytical and instrumental errors
 To increase trust of laboratory customers
 To ensure the quality of the test results
 To meet accreditation requirement

6. ANALYTICAL CHARACTERISTIC IN ANALYTICAL METHOD VALIDATION


1. Linearity
 Linearity of an analytical procedure as the ability to obtain test results of variable data which
are directly proportional to the concentration of analyte in the sample.
2. Selectivity/Specificity
 The ability of the method that can determine a particular compound in the matrices without
interference from matrix components (e.g., impurities, degradation products and matrix
components).
→ spiking samples
3. Limit of Detection
 The lowest concentration of analyte in a sample that can be detected, but not necessarily
quantitated under the stated conditions of the test.
 Procedure for MDL
o Analyse 10 independent sample blanks measured once each
o Calculate standard deviation (s) of sample blank
o Calculate mean value of sample blank
4. Limit of Quantitation
 LOQ is the lowest concentration of an analyte that can be determined with acceptable
precision (repeatability) and accuracy under the stated conditions of the test.
 Procedure for LOQ
o Analyse 10 independent sample blanks measured once
o Calculate standard deviation (s) of sample blank
o Calculate mean value of sample blank
5. Precision
 The closeness of agreement between independent test results obtained under stipulated
conditions. (How close results are to one another).
 Three types of precision measurement:
o Repeatability
Precision under repeatability conditions, i.e. conditions where independent test
results are obtained with the same method on identical test items in the same laboratory
by the same operator using the same equipment within short interval of times.
o Intermediate precision (internal reproducibility)
Precision measured between analyses, over extended timescale, within a single
laboratory
o Reproducibility
Precision under reproducibility conditions, i.e. conditions where independent test
results are obtained with the same method on identical test items in different
laboratories with different operator using different equipment. Proficiency
testing/collaborative studies between laboratories.
6. Recovery
 Recovery is the ratio of the observed value to the expected
 value.
 Recovery is evaluated:
o by analysis of a Certified Reference Material (CRM), or
o by fortified test portion with analyte and measured the analyte concentration.
7. Robustness
 Intra-laboratory study to check changes due to environmental and/or operating conditions
 Procedure
o Carry out at least 6 replicate analyses on a sample using the analytical method under
validation with some critical conditions such as:
 Small change of pH level
 Extraction time
 Temperature of extraction
 Change of flow rate
 Change of another volatile solvent

7. WHEN SHOULD METHODS UNDERGO VALIDATION


 New method development
 Revision of established methods
 When established methods are used in different laboratories/different analysts etc.
 Comparison of methods

8. WHEN SHOULD METHODS UNDERGO VERIFICATION


 Major instruments are replaced
 Methods are used for the first time by a new staff
 The laboratory takes an already validated and verified method into use after it has not been used for
a long time

9. STATE THREE APPLICATIONS OF SOXHLET EXTRACTION - lab


I. Pharmaceutics
II. Environment
III. Foodstuffs

10. WHEN DO WE USE HYDROFLUORIC ACID OR PERCHLORIC ACID FOR DIGESTION? - lab
Hydrofluoric acid is used in industry to digest to digest silicates in ore samples. In organic sample digestion,
nitric acid is rarely used alone. It is best used in the combination with sulphuric and/ or perchloric acid for
organic sample digestion. For samples that are not highly aromatic and/or contain a high -OH functionality,
nitric acid is preferably used followed by perchloric acid. The only element that may be lost from a
nitric/perchloric digestion is Hg

11. FUNCTION NITRIC ACID – test, lab


Most heavy metals exist in combination of other organic materials in soil or plant matter, therefore acid
help in breaking existing bonds between the metals and the matrix in which they are to be extracted from.
Most metals however require acid digestion for solubility purpose

12. DISCUSS THE FUNCTION OF USING ACID HYDROCHLORIC ACID AND NITRIC ACID DURING THE
DRY ASHING PROCESS - lab
The function of acids in dry ashing is to dissolve the residue. Concentrated hydrochloric acid dissolved
many metals and forms oxidised metal chlorides and hydrogen gas. HCl is used in order to make some
organic compounds soluble in water. This compounds go through reactions to take in the chloride ion.
This makes the organic compound more polar by introducing a highly electronegative atom, which is
chlorine. Nitric acid can oxidise non-active metals
13. SOXHLET VS SHAKE FLASK EXTRACTION

SOXHLET EXTRACTION DIFFERENTIATE SHAKE FLASK EXTRACTION


Solvent reservoir, extraction body,
heat source (e.g. isomantle) and APPARATUS USE Stoppered glass container
water cooled reflux condenser
In the inner tube of soxhlet
SAMPLE LOCATED Into suitable glass container
apparatus
Sample plus dispersion agents placed
in porous extraction thimble and EXTRACTION Adding suitable organic solvent and
heat applied via isomantle (4 cycles TECHNIQUE the agitating shaking (twice)
per hour)
Solvent containing the analytes is AFTER COMPLETION
All solvent extracts combined
retained OF EXTRACTION

14. HPLC VS GC
 Non-volatile compounds can be easily analysed on HPLC whereas GC is useful for analysis of
volatile compounds
 In GC, the mobile phase is a gas whereas in HPLC the mobile phase is liquid.
 HPLC column are shorter and wide in comparison to GC column.
 HPLC requires higher operating pressures than GC because liquids require higher pressures
than gases for transport through the system.
 HPLC is useful for analysis of samples, which decompose at higher temperatures.
 HPLC can be used for analysis of very high molecular weight compounds.

CRITERIA HPLC GC
 Use liquid as carrier
 Use gas as carrier
MOBILE PHASE  Mixture of solvent of compatible
 Single high purities gas
polarities
 Separations carried out at
 Separation carried out at ambient
elevated temperature
OPERATING temperature
 Can be held at constant value
TEMPERATURE  Packing shave extended temperature
or variables as decided to temp.
limit
program
 Compound ranging in molecular
 Compound separated have higher
weight up to few hundred
NATURE OF molecular weight ranging from a few
 Such compound separate on
COMPOUNDS hundred to several millions for large
differences in their volatilities &
polymer & biomolecules
remain stable at high temp.
COLUMN  Much longer & narrower
 Much shorter & wider dimension
DIMENSION dimension
 Higher permeability of solid
 Offer greater resistance to flow of
supports than liquid
COLUMN liquids
 The retention mode depends
PACKING  The retention mode depends on
on difference in volatilities of
polarity differences/molecular size
compounds
 Based on non-destructive detection
DETECTION such as UV, EI, photodiode array  Based on destructive principle
PRINCIPLE detectors, conductivity, laser such as FID, NPD, FPD
detection
 Solvent are costly
 Maintenance cost of HPLC system  Solvent less costly
COST OPERATION
higher due to pressure develop in  Less maintenance cost
column & pump
 Mobile phase
o As the name suggests High Performance Liquid Chromatography uses a liquid mobile phase and
gas chromatography uses a gas as the carrier. Liquids are generally mixtures of solvents of
compatible polarities whereas in gas chromatography the mobile phase is a single high purity
gas.
 Operating temperature
o HPLC separations are mostly carried out at ambient temperatures whereas Gas
Chromatography separations are carried out at elevated temperatures which can be held at a
constant value (isothermal) or variable as decided by the temperature program. Newer packings
have extended temperature limits of High Performance Liquid Chromatography operation as
well.
 Nature of compounds
o Gas chromatography separations are mainly carried out on compounds ranging in molecular
weights up to a few hundreds. Such compounds separate on differences in their volatilities and
remain stable at high temperatures. On the other hand compounds separated on HPLC have
higher molecular weights ranging from a few hundreds to several millions for large polymers and
biomolecules. Such compounds can be analysed at room temperature only because at elevated
temperatures they tend to degrade.
 Column Dimensions
o Liquids used as carrier in HPLC generally have higher viscosity in comparison to gases used in Gas
Chromatography. This results in increased column back pressures in HPLC. It is for this reason
that High Performance Liquid Chromatography columns are much shorter and have wider
diameters in comparison to GC columns which can be much longer and narrower. Increased
column length improves resolution between closely spaced peaks. As the trend is towards faster
analysis columns used for HPLC are as short as 1 cm in length.
 Column Packings
o Column packings offer greater resistance to flow of liquids in comparison to gases. Gases also
have a higher permeability of solid supports than liquids. The retention mode of HPLC columns
depends on polarity differences or molecular sizes whereas GC separations are based on
differences in volatilities of compounds
 Detection principles
o HPLC detection is commonly based on non-destructive detection such as UV, RI, photodiode
array detectors, conductivity and laser detection. On the other hand, Gas
Chromatography detection is based largely on destructive principles such as a FID, NPD and FPD.
Mass spectrometry detectors common to both LC and GC are destructive in nature.
 Cost of operation
o HPLC solvents are costly in comparison to gases used for GC analysis. In addition to cost of
solvents the maintenance cost of High Performance Liquid Chromatography systems is also
higher due to high pressures developed in pumps and columns. GC analysis in comparison is less
costly and there is lesser maintenance cost.

15. MICROWAVE DIGESTION SYSTEMS OFFER SEVERAL DISTINCT ADVANTAGES OVER OPEN
DIGESTIONS
 Biggest benefit is time saving. Simultaneous heating of 8 – 12 samples is possible and reaction times
are typically less than an hour in comparison to 5 – 12 hours or even more for open digestions.
 Time taken for a digestion is dependent on temperature. Open digestions are limited to boiling points
of acids used whereas closed digestion systems can be operated at higher temperatures of 250
– 300OC
due to ability to withstand higher pressure.
 Acid consumption is lower in microwave digestion
 Microwave digestion systems offer greater extraction efficiency whereas in open system digestion
extraction can be incomplete depending on discretion of the analyst.
 No exposure of analyst to corrosive acid fumes as compared open digestions
 Loss of volatile elements such as Hg or Pb can take place in open digestion whereas in closed system
digestions loss of volatile elements is prevented.
 A greater risk of contamination from external sources exists in open digestion whereas in closed
systems the risk is non-existent

Closed microwave digestion systems have only one disadvantage, namely, explosion and cracking of digestion
tubes due to simultaneous build – up of pressure along with increase of temperature. However, microwave
systems have inbuilt sensors for both temperature and pressure. The microwave power is automatically
controlled or shut-off when pressure reaches the maximum limit

16. PROCEDURE LLE – test


17. EXCITATION AND RELAXATION OF MOLECULE - test
18. DIFFERENTIATE BETWEEN ULTRASONIC EXTRACTION AND MICROWAVE ASSISTED
EXTRACTION
19. BENEFITS OF SOLID PHASE MICRO EXTRACTION
20. COLLECTION METHOD OF VOC
21. TWO ATTRIBUTES/PROPERTIES/FUNCTION OF INFRARED SPECTROSCOPY
22. FUNCTION OF NEBULIZER OF AAS - test
23. ION EXCHANGE CHROMATOGRAPHY
24. THE SOLUTION RISE UP THE ANALYTE TO THE COLUMN OF HPLC
25. ISOCRATIC MOBILE PHASE AND GRADIENT MOBILE PHASE
26. TWO FACTORS RELATED TO HPLC/GC/POLARITY&POLARITY/STATIONARY PHASE AND MOBILE
PHASE
27. WHAT IS SOXHLET EXTRACTION? - lab
28. EXPLAIN THE PRINCIPLE OF SOXHLET EXTRACTION - lab
29. DOES METHYL MERCURY AND INORGANIC MERCURY CAN BE CARRIED OUT USING SAME
EXTRACTION PROCEDURE