TECHNIQUES & PROTOCOLS FOR INSTRUMENTATION ANALYSIS (HPLC & CENTRIFUGATION MACHINE)

TECHNIQUES & PROTOCOLS
FOR INSTRUMENTATION
ANALYSIS
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC)
&
CENTRIFUGATION MACHINE
2

Table of Contents
1. Instrumental Analysis
2. Techniques & Protocol
3. What Is Method Validation?
4. Steps In The Chromatographic Methods Validation Process
5. High Performance Liquid Chromatography (HPLC)-principle
6. Characterization Of HPLC
7. Constituents Of HPLC
8. Method Development In HPLC
3

Table of Contents…
9. Strategy For Method Development In HPLC
10. Instrumentation Of HPLC System
11. Calibration Of HPLC Instrument
12. Separation Mode
13. Maintenance
14. Troubleshooting
15. Applications Of HPLC
4

INSTRUMENTAL ANALYSIS
Instrumental analysis is a field of analytical
sciences that investigates instruments using scientific
principles.
Basics of Instrumental analysis:
1. Interpretation of data obtained from measurements
2. Communication of meaning of the results
5

 TECHNIQUES:
Chemical or physical principle we can use
to study an Instrument
 PROTOCOL:
Set of stringent guidelines specifying a
procedure
TECHNIQUES & PROTOCOL
6

What is Method Validation?
Guidance for industry on analytical procedures and methods
validation , states:
“Methods validation is the process of
demonstrating that analytical procedures are
suitable for their intended use. The methods
validation process for analytical procedures begins
with the planned and systematic collection by the
applicant of the validation data to support
analytical procedures”
1. Undergraduate Instrumental Analysis, James W. Robinsosn 7

Steps in the chromatographic methods
validation process
The Process of validating chromatographic methods can be broken
down into four steps. These steps include:
1. Method evaluation and further method development
2. Final method development and trial methods validation.
3. Formal methods validation, and
4. Formally data review and report issuance.
2. Undergraduate Instrumental Analysis, James W. Robinsosn 8

HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC)
Principle:
“A liquid mobile phase is pumped under pressure
through a stainless steel column containing particles of stationary
phase with a diameter of 3-10µm (1.7µm in Ultra high
performance liquid chromatography (UPLC)). The analyte is
loaded onto the head of the column via a loop valve and
separation of mixture occurs according to relative lengths of time
spent by its components in a mixture spend more or less the same
time in the mobile phase in order to exit the column. Monitoring
of column effluent can be carried out the variety of detectors”.
3. Pharmaceutical Analysis David G. Watson 9

CHARACTERIZATION OF HPLC
HPLC is characterized by the use of high pressure to push a
mobile phase solution through a column of stationary phase
allowing separation of complex mixtures with high
resolution.
4. Pharmaceutical Analysis David G. Watson 10

CONSTITUENTS OF HPLC
 Mobile Phase:
The eluate moving through the column.
 Stationary Phase:
The substance that remains in one place in the column.
 Eluate:
The mobile phase exiting a column.
 Eluent:
The mobile phase entering a column.
 Elution:
The passage of the mobile phase through the column
 Column:
To transport solutes
5. High-Throughput Analysis in the pharmaceutical industry, Perry G. Wang 11

Method Development in HPLC
“Best column, best mobile phase, best detection wavelength, efforts
in their selection can make a world of difference while developing
HPLC method for routine analysis. Determining the ideal
combination of these factors assures faster delivery of desired
results----a validated method for separation.”
• On sample properties as much knowledge as possible should
be collected.
• Chromatography method is selected according to the sample
properties.
• The sample is chromatographed with HPLC conditions
where all compounds elute in a reasonable time.
• The HPLC method is optimized with regard to analysis time
resolution, selectivity and sensitivity.
6. High Performance Liquid Chromatography Fundamental Principles and Practice W.J. Lough 12

Strategy for method development in HPLC
Selection of a suitable chromatography method for organic
compounds:
• First Reversed-phase should be tried.
• If not successful, Normal-phase should be taken into
consideration.
 Before making experiments with ION-exchange or Ion-pair
chromatography, first ion suppression by pH control and
reversed-phase chromatography should be tried.
 For ion-forming organic compounds Ion-pair chromatography
should be preferred ion-exchange chromatography.
7. High Performance Liquid Chromatography P.D. Sethi 13

Selection of Chromatographic Method
14

A modern self-contained HPLC
8. Validating Chromatographic Methods, A practical guide David M. Bliesner 15

Schematic representation of HPLC
(1) Solvent reservoirs, (2) Solvent degasser, (3) Gradient valve, (4) Mixing vessel
for delivery of the mobile phase, (5) High-pressure pump, (6) Switching valve in
"inject position", (6') Switching valve in "load position", (7) Sample injection loop,
(8) Pre-column (guard column), (9) Analytical column, (10) Detector (i.e. IR,
UV), (11) Data acquisition, (12) Waste or fraction collector.
16

Instrumentation of HPLC system
1. Solvent (Mobile Phase)
2. Degasser
3. Solvent Delivery System (Pump)
4. Sample Injector
5. Column
6. Stationary Phase
7. Detectors (Diode Array)
8. Waste Collector
9. Recorder (Data Collection)
9. Chromatographic methods 5th Edition By A. Braithwaite & F.J. Smith
17

1. Mobile Phase in HPLC
o Compatible with the instrument (pumps, seals, fittings,
detector, etc)
o Compatible with the stationary phase
o Readily available (often use liters/day)
o Of adequate purity
o Not too compressible (causes pump/flow problems)
o Free of gases (which cause compressibility problems)
10. Quantitative Chemical Analysis 7th Edition Daniel C. Harris 18

2. Degasser
This step is done to remove the air bubbles present
in the soluble form in the mobile phase.
Procedure:
For the removal of the air we have required inert
gas for example argon, helium, nitrogen. They take the place of
air and are lighter as compared to the soluble air. In other way
they create a vacuum over M.P. Through vacuum the air or any
gasses present in the mobile phase come out.
11. Chemical Analysis Modern Instrumentation Methods and Techniques 2nd Edition Francis Rouessac and Annick
Rouessac
19

3. Pumps
Ideal properties of pump
o It must have the ability to generate the pressure of 6000 psi.
o Non-pulsative delivery of the mobile phase (steady flow).
o Flow rate must between 0.1-10 ml/min.
o Non-corrosive to the different solvents used in HPLC.
12. VOGES Textbook Of Quantitative Chemical Analysis By MENDHAM
20

4. HPLC Chromatograph injectors
• The function of the injector is to place the sample into the
high-pressure flow in as narrow volume as possible so that the
sample enters the column as a homogeneous, low-volume plug.
• To minimize spreading of the injected volume during transport
to the column, the shortest possible length of tubing should be
used from the injector to the column
13. Instrumental Techniques For Analytical Chemistry By FRAUT SELLER 21

5. Columns of HPLC
o The column is one of the most important components of the
HPLC chromatograph because the separation of the sample
components is achieved when those components pass through
the column.
14. A Textbook Of Analytical Chemistry Mahinder Singh 22

6. Stationary Phase
o Normally, columns are filled with silica gel because its particle
shape, surface properties, and pore structure help to get a good
separation.
o Silica is wetted by nearly every potential mobile phase, is inert to
most compounds and has a high surface activity which can be
modified easily with water and other agents
15. Chromatography CONCEPTS & CONTRASTS By James M. Miller
23

7. Detection in HPLC
o The chromatographic detector is capable of establishing both the
identity and concentration of eluting components in the mobile
phase stream.
o A broad range of detectors is available to meet different sample
requirements.
o Specific detectors respond to a particular compound only and the
response is independent of mobile phase composition.
16. Quantitative Chemical Analysis 7th Edition Daniel C. Harris 24

CALIBRATION OF HPLC INSTRUMENT
Calibration of HPLC is done to check the performance of its
instrument.
1. Flowrate (pump calibration)
2. Detector and injector linearity
3. System precision
4. Column oven temperature
5. Detector wavelength accuracy
17. High Performance Liquid Chromatography P.D. Sethi 25

1. PUMP CALIBRATION
• Disconnect the column and connect the inlet and outlet
tubing’s with a union.
• Prime all the lines at 5 ml/min flow rate with water and
ensure that flow line is free from air bubbles.
• Set the flow rate at 1ml / min and collect the mobile phase
(water) in a dry Preweighed beaker and collect the mobile
phase for 10 min. weigh the beaker to get the weight of
mobile phase.
18. High Performance Liquid Chromatography Fundamentals Principles P.D. Sethi 26

2.DETECTOR AND INJECTOR LINEARITY
• Column : C18
• Mobile phase : milli Q water and acetonitrile (80:20)
• Flow rate: 1ml/min
• Temperature: 40 Centigrade
• Detector wavelength:272 nm
• Runtime:10min
19. High Performance Liquid Chromatography Fundamentals Principles P.D. Sethi
27

3. SYSTEM PRECISION
Create and instrument method with a wavelength in nm and
inject blank followed by Standard preparation and note down
the height or absorbance.
Acceptance criteria: The maximum absorbance should be ±2nm
28

4. COLUMN OVEN TEMPERATURE
• Set the column oven temperature to 30 and leave it for 30
minutes
• Open the door of the column oven and keep the thermometer
and leave it for 30 min
• Now note down the reading in the thermometer
• Similarly change the column oven temperature to 40C and 50C
and repeat the above procedure.
29

5.DETECTOR WAVELENGTH ACCURACY
• Create and instrument method with a wavelength in nm and
inject blank, followed by Standard preparation and note down
the height or absorbance.
• Acceptance criteria: The maximum absorbance should be
±2nm
30

Separation ModeSeparation in based upon
differential
migration between the stationary
and mobile phases.
Injector
Mixer
Pumps
Column
Detector
Waste
High Performance Liquid Chromatograph
52
Solvents
Mobile Phase - carries the sample
through the stationary phase as it
moves through the column.
Stationary Phase - the phase
which remains fixed in the
column, e.g. C18, Silica
31

Separations
Injector Chromatogram
Mixer mAU
Pumps
timeStart Injection
Column
Detector
Solvents
High Performance Liquid Chromatograph
53 32

Mixer mAU
Pumps
timeStart Injection
Column
Detector
Solvents
54
Separations
33

Mixer mAU
Pumps
timeStart Injection
Column
Detector
Solvents
55
Separations
34

Mixer mAU
Pumps
timeStart Injection
Column
Detector
Solvents
56
Separations
35

Mixer mAU
Pumps
timeStart Injection
Column
Detector
Solvents
57
Separations
36

Mixer mAU
Pumps
timeStart Injection
Column
Detector
Solvents
58
Separations
37

Mixer mAU
Pumps
timeStart Injection
Column
Detector
Solvents
59
Separations
38

Mixer mAU
Pumps
timeStart Injection
Column
Detector
Solvents
60
Separations
39

Mixer mAU
Pumps
timeStart Injection
Column
Detector
Solvents
61
Separations
40

Mixer mAU
Pumps
timeStart Injection
Column
Detector
Solvents
62
Separations
41

Mixer mAU
Pumps
timeStart Injection
Column
Detector
Solvents
63
Separations
42

Mixer mAU
Pumps
timeStart Injection
Column
Detector
Solvents
64
Separations
43

Mixer mAU
Pumps
timeStart Injection
Column
Detector
Solvents
65
Separations
44

Mixer mAU
Pumps
timeStart Injection
Column
Detector
Solvents
66
Separations
45

Mixer mAU
Pumps
timeStart Injection
Column
Detector
Solvents
67
Separations
46

Mixer mAU
Pumps
timeStart Injection
Column
Detector
Solvents
68
Separations
47

MAINTAINANCE
1.RESERVOIR
Possible Cause Preventive Maintenance
Blocked inlet frit
o Replace (3–6 months)
o Filter mobile phase with 0.4 - 0.5 μm
filter
Gas bubbles Degas mobile phase, sonification
48

MAINTENANCE
Air Bubbles Degas mobile phase , do not change mobile
phase during run
Pump Seal Failure Replace (3 months),clean with 1 N acid
Check valve
failure
Filter mobile phase; use inlet line frit ; keep
spare
Improper cleaning Clean with Isopropyl alcohol , mobile phase
container must be cleaned with mobile
phase and other sections with solvent
2. PUMP
49

MAINTAINANCE
3.INJECTOR
Washing wash before and after use
Rotor seal wear Do not overtighten
Syringe Sterilize when fresh sample is used
50

MAINTAINANCE
Number of
injections
2000 or less
Blocked frit o Filter mobile phase
o Filter samples
o Use in-line filter and/or guard Column
Void at head of
column
o Avoid mobile phase pH >8
o Use guard column
o Use precolumn
4.Column
51

MAINTAINANCE
5.Detector
Lamp failure Replace (6 months) or keep spare lamp
Bubbles in cell o Keep cell clean
o Use restrictor after cell
o Degas mobile phase
52

MAINTAINANCE
6. Software
Update frequently , around 6-12 months
53

Troubleshooting
There are five major of symptoms to help you quickly identify the
source of the problem(s):
I. Pressure abnormalities
II. Leaks
III. Problems with the chromatogram
IV. Injector problems
V. Other problems detected by the senses of smell, sight, and
sound, when you have corrected the problem, record the incident
in the system record book to help with future problems.
54

Troubleshooting
III- PROBLEMS WITH THE CHROMATOGRAM
Problem-1: Peak tailing
1. Block Frit Replace inlet frit
2. Column Void Fill void
3. Wrong mobile phase pH Adjust pH
55

Troubleshooting
Problem-2: Peak fronting
1. Low temperature Increase column temperature
2. Sample overload Decrease sample concentration
3. Wrong sample solvent Use mobile phase for injection
solvent
56

Troubleshooting
Problem-3: Split peaks
1. Contamination on guard and analytical column inlet
“Remove guard column and replace if necessary. If analytical
column is obstructed, Change frit or replace column”
57

Troubleshooting
Problem-4: Baseline Drift
1. Column temperature Control column & mobile
phase temperature
2. Mobile phase mixing
problem or change in flow rate Correct composition / flow
rate
58

Troubleshooting
Problem-5: Baseline Noise (irregular)
1. Leak Check system for loose fittings
2. Weak detector lamp Replace lamp
3. Air trapped in system Flush system with strong solvent
59

Troubleshooting
1. M.P Flow rate too slow Adjust M.P flow rate
2. Guard column contaminated Replace guard column
/worn out
Problem-6: broad peak
Causes
60

Applications of HPLC
HPLC is one of the most widely applied analytical separation
techniques.
Pharmaceutical:
• Tablet dissolution of pharmaceutical dosages.
• Shelf life determinations of pharmaceutical products.
• Identification of counterfeit drug products.
• Pharmaceutical quality control.
61

Environmental:
• Phenols in Drinking Water.
• Identification of diphenhydramine in sediment samples.
• Biomonitoring of pollution in high-altitude mountain lakes
through the analysis of fish bile.
Forensics:
• A mobile HPLC apparatus at dance parties - on-site
identification and quantification of the drug Ecstasy.
• Identification of anabolic steroids in serum, urine, sweat and
hair.
• Forensic analysis of textile dyes.
62

Clinical:
• Quantification of DEET (diethyltoluamide) in Human Urine.
• Analysis of antibiotics.
• Detection of endogenous neuropeptides in brain extracellular
fluids.
63

TECHNIQUES & PROTOCOLS
FOR INSTRUMENTATION
ANALYSIS
CENTRIFUGATION MACHINE
64

PRINCIPLE
65
“The centrifuge using the sedimentation principle, where the
centripetal acceleration causes denser substances and particles to
move outward in the radial direction. At the same time objects
that are less dense are displaced and move to the center”.
Principles and technique in biochemistry-L WALKER & WILSON

Purpose of Equipment calibration
66
• To determine the accuracy and precision of the parameters of
an equipment in relation to its use.
• To determine the uncertainty of the parameters within a
stated confidence level (usually 95%).
• To maintain the traceability of the results provided by the
equipment
Lynch's Medical Laboratory Technology

Parameters of an instrument to be calibrated
67
• Any parameter of an instrument that will affect the
quality of the products/test results has to be
calibrated.
(e.g., a refrigerated centrifuge : speed, time,
temperature)

General Requirements of Equipment
Calibration
68
• All equipment must be uniquely identified and labelled.
• Acceptance criteria should be set for each type of equipment
• All equipment should be calibrated prior to being placed into
service, recalibrated on schedule and after major repair.
• Equipment calibration method must meet the accuracy and
precision requirements of the equipment.

Centrifuge Calibration
69
Purpose:
This procedure provides accurate rotation speed, timer
verification and centrifuges that are temperature controlled in a
laboratory environment.

Verification of Timer
70
• Centrifuges used in the laboratory are to be considered as
contaminated and should only be handled with gloves and
other personal protective equipment and/or thoroughly
disinfected before calibration verification.
• Set the centrifuge timer at a setting frequently used in
procedures, and start the stopwatch simultaneously.
• Stop the stopwatch at the same time as the centrifuge timer
ends.
• Calculate the difference between the two times .

Verification of rotation speed
71
• Centrifuges used in the laboratory are to be considered as
contaminated and should only be handled with gloves and other
personal protective equipment and/or thoroughly disinfected
before calibration verification.
• Place a small section of black and white reflective tape that
comes with the tachometer on the center spindle of the test
centrifuge.
• In order to measure the rotation speed, there must be a viewing
port in the top cover that will allow the tachometer line of sight
to the reflective tape

72
• Place a normal well-balanced load using specimen covers into
the centrifuge.
• Start the centrifuge and allow it come to equilibrium at a
normal operating speed.
• Use the tachometer through the viewing port above the
reflective tape to take a rotation rate reading.
• Record the rotation speed indicated by the centrifuge either by
the dial setting or by a built in tachometer on the centrifuge .
Verification of rotation speed…

73
Calibration of Centrifuge Speed

Calibration of temperature
74
It includes:
A) Fixed angle rotor:
B) Swing out rotor

Calibration of temperature…
75
A) Fixed angle rotor:
• Fill the two sample tubes with ethylene glycol and place it in
the sample holder. Set the parameters like temperature 5°C,
RPM: 12000 and time 45 minutes.
• Record the temperature of the sample solution by using digital
thermometer.

Calibration of temperature…
76
B) Swing out rotor
• Fill the two sample tubes with ethylene glycol and place it in
the sample holder. Set the parameters like temperature 5°C, RPM:
4000 and time 45 minutes.
• Record the temperature of the sample solution by using digital
thermometer.

Interpretation of results
77
Verification of rotation speed:
• If the difference between the test centrifuge and the certified
tachometer is ± 5% of the procedure speed, then the test
centrifuge rotation calibration is verified as acceptable.
Verification of timer:
• Acceptable difference between the test timer and the certified
timer must be ±2% of the total test timer setting.

Calibration Report for Centrifuge
78

Maintenance of Centrifuge
79
Daily maintenance:
• Wipe the inside of the bowl with disinfectant solution and rinse
thoroughly.
• The centrifuge must not be used if the interior is hot, if unusual
vibrations or noises occur, or if deterioration (corrosion of
parts) is detected.
• A qualified service technician should be contacted.
• Most vibrations are due to improper balancing and can be
corrected by rebalancing the buckets and tubes.

80
Monthly maintenance:
• Clean the centrifuge housing, rotor chamber, rotors and rotor
accessories with a neutral cleaning agent.
• Clean plastic and non-metal parts with a fresh solution of
0.5% sodium hypochlorite .
Maintenance of Centrifuge…

81
• Remove cellular elements from blood to provides cell free serum
or plasma.
• Conc. Cellular element for microscopy.
• Remove protein precipitate from analytic sample.
• Isolation of macromolecules such as DNA, RNA, proteins, or
lipids.
• Use in hematology lab for PCV determination.
Application

82
Thank you for your attention, I hope this mesmerises
you enough to prevent you from asking difficult
questions!!!

TECHNIQUES & PROTOCOLS FOR INSTRUMENTATION ANALYSIS (HPLC & CENTRIFUGATION MACHINE)

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