Series:

“Let’s
Educate
Each
Other”
Session 1: “HPLC System Calibration”
Presenter: Dr. Ravi Kant Rajpoot, Ph.D.
 What is Calibration and Why it is Important?
• What is Calibration?

- Calibration is a documented evidence that the HPLC system is working properly and is suitable for its intended
purpose.

• Why is it Important?

- HPLC machine is a high throughput system that is widely used for routine analysis in pharmaceutical/
biopharmaceutical industries. So, it is a must that it should provide accurate and reliable results.
- Calibration of an HPLC machine is necessary to give quantitative results.

- ISO/IEC 17025, General requirements for the competence of testing and calibration laboratories, is the
international reference for testing and calibration laboratories wanting to demonstrate their capacity to deliver
reliable results. It mandates periodic calibration of HPLC systems.

- Regulatory agencies, such as the FDA, require that analytical methods (HPLC based) used in research and
manufacturing must be validated on calibrated HPLC systems to verify the quality and safety of
pharmaceutical products.

- For ensuring product quality, HPLC systems should demonstrate reproducibility of results and should be able to
detect any potential deviations in product quality.
- Accurate analysis using HPLC is critical in determining the quality, purity, and potency of the products, which
directly impacts patient safety.
<USP 1058> ANALYTICAL INSTRUMENT QUALIFICATION

- A large variety of analytical instruments, ranging from a simple apparatus to complex computerized systems, is
used in the pharmaceutical industry to acquire data that will help ensure that products meet their specifications.

- The risk assessment for an Analytical Instrument Qualification (AIQ) enables the classification of the
instrument to determine the extent of qualification and actions needed to demonstrate fitness for purpose.

- Instruments can generally be classified as belonging to Groups A, B, or C. It should be noted that the same
type of instrument can fit into one or more categories, depending on its intended use.

- Group A includes the least complex, standard instruments that are used without measurement capability or
user requirement for calibration, such as a magnetic stirrer or vortex mixer. Proper function is ensured by
observation, and no further qualification activities are needed for this group.

- Group B includes instruments that may provide a measurement or an experimental condition that can affect
a measurement. Examples include a pH meter or an oven. Proper function of instruments in this group may
require only routine calibration, maintenance, or performance checks. The extent of activities may depend
on the criticality of the application. Generally, these instruments may have firmware but not software that is
updated by the User.
- Group C comprises analytical instruments with a significant degree of computerization and complexity, such as
high-pressure liquid chromatographs and mass spectrometers. All elements of qualification, including
software validation, must be considered to ensure proper functioning of instruments in this group.
<USP 1058> ANALYTICAL INSTRUMENT QUALIFICATION

Groups of Instruments as per <USP 1058>

Group A Group B Group C
Magnetic stirrers Balances High-pressure liquid chromatographs
Vortex mixers Variable pipettes Mass spectrometers
Nitrogen evaporators pH meters Microplate readers
Centrifuges Refractometers Electron microscopes
Viscometers Differential scanning calorimeters
Melting point apparatus Atomic absorption spectrometers
Thermometers Gas chromatographs
Titrators Raman spectrometers
Light microscopes UV/vis spectrometers
Ovens Atomic absorption spectrometers
Refrigerator freezers Diode-array detectors
Pumps Densitometers
Water baths Near-IR spectrometers
 Arc and Alliance HPLC Systems Specifications
Spec Arc HPLC System Alliance HPLC System
SYSTEM FEATURES
Operating flow 0.001 to 5.000 mL/min, in 0.001 mL increments 0.01 to 10.000 mL/min (0.050 to 5.000 mL/min
typical) in 0.001 mL/min increments
rate range
pH range 1 to 12.5 Not found in search
QUATERNARY SOLVENT MANAGER-R
Blend up to four solvents in any combination Blend one to four solvents in any combination
Solvent capacity
(standard)
Gradient
Low-pressure mixing, Quaternary gradient Quaternary gradient
formation
± 1.0% at 0.5, 3.0, and 5.0 mL/min ±1% or 10 μL/min, whichever is greater, 0.200 to
Flow accuracy
5.000 mL/min
≤0.075% RSD or ± 0.020 min SD, whichever is ≤0.075% RSD or ≤0.02 min SD, six replicates,
Flow precision
greater, based on six replicates. based on retention time or volumetric measures
(0.200 to 5.000 mL/min)
(Flow precision is critical for repeatability of peak
height and area)
≤0.5 mAU [mobile phase containing 0.1% TFA in Not found in search
Composition
water/acetonitrile; 1.5 mL/min; CORTECS C18 2.7
ripple
μm, 4.6 x 50 mm; 35 °C; UV @214 nm]
± 0.5% absolute (full scale) from 5 to 95%; 0.5 to ± 0.5% absolute, independent of backpressure
Composition 5.0 mL/min [methanol; methanol with 5.0 mg/mL (proportioning valve pair test, [degassed
accuracy caffeine step gradient; UV @273 nm] methanol:methanol/propylparaben, 2.0 mL/min,
254 nm])
 Arc and Alliance HPLC Systems Specifications
Spec Arc HPLC System Alliance HPLC System
QUATERNARY SOLVENT MANAGER-R (Contd.)
± 0.15% RSD or 0.04 min SD, whichever is greater ≤0.15% RSD or ≤0.02 min SD, whichever is
based on six replicate injections [60:40 greater, based on retention time (60:40
Composition water:methanol via Auto•BlendTM Technology; 0.5 degassedmethanol/water dial-a-mix, 1.00 mL/min,
precision mL/min; alkylphenone mix; 24.0 μL injection six replicates, phenone mix, 254 nm)
volume; CORTECS C18 2.7 μm, 4.6 x 50 mm; 35
°C; UV @254 nm]
316L stainless steel, PPS, fluoropolymer, 316 stainless steel, ruby, sapphire, MP35N, PEEK,
Primary wetted
UHMWPE blend, sapphire, ruby, zirconia, DLC, PPS, UHMWPE, Tefzel (ETFE), Teflon (FEP and
materials
PEEK and PEEK blend, titanium alloy PTFE), Teflon AF, Fluoroloy G, Fluoroloy-08R
SAMPLE MANAGER FTN-R
Injection volume 0.1 to 50.0 μL as standard 0.1 to 100.0 μL as standard
range
Any two of the following: 48-position, 2.00-mL vial 120 vials, configured in five carousels of 24 vials
Sample capacity holder (total capacity of 96 vials), 96-well plate, each
384 well plate, etc.
Sample 4.0–40.0 °C, settable in 0.1 °C increments; 4 to 40 °C, programmable in 1 °C increments
compartment Temperature accuracy: ± 0.5 °C at the sensor Temperature accuracy: ± 2 °C at the sensor
Temperature Temperature stability: ± 1.0 °C at the sensor Temperature stability: ± 1.0 °C at the sensor
Minimum 3 μL residual, using total recovery 2-mL vials 10 μL, using low volume inserts
sample required
Accuracy ± 0.2 μL Not found in search
(aspiration)
>0.999; 0.2–50.0 μL >0.999 coefficient of deviation (1.000 to 100.000
Linearity
μL)
 Arc and Alliance HPLC Systems Specifications
Spec Arc HPLC System Alliance HPLC System
SAMPLE MANAGER FTN-R (Contd.)
<1.0% RSD from 0.5 to 0.9 μL <0.5% RSD from Typically <0.5% RSD, 5 to 80 μL
Precision
1.0 to 4.9 μL <0.25% RSD from 5.0 to 1000.0 μL Typically <0.3% RSD, 5 to 60 μL
Sample ≤0.002% [Caffeine] under UV conditions Sample carryover ≤0.0025% for caffeine, under
carryover specified conditions
Primary wetted 316L stainless steel, gold plated stainless steel, —
materials polyimide, PEEK blend, DLC
30-CM COLUMN HEATER AND HEATER COOLER (30-CM CH AND 30-CM CHC)
Single column, up to 7.8 mm I.D.; up to 300 mm
length with filter or guard column; up to three
Column capacity
columns with optional 3-position column selection
valve
Column 30-cm CHC: 4.0 (or 15.0 °C below ambient, Ambient minus 15 or 4 °C (whichever is greatest)
compartment whichever is greater) to 65.0 °C up to 65 °C, in 1 °C increments
temperature
+/- 0.5 °C at the sensor
Temperature
30-cm CH: 20.0 (or 5.0 °C above ambient) to 65.0
accuracy
°C
Temperature +/- 0.3 °C at the sensor
stability
 Arc and Alliance HPLC Systems Specifications
Spec Arc HPLC System Alliance HPLC System
ENVIRONMENTAL SPECIFICATIONS
Operating temperature range 4 to 40 °C 4 to 40 °C
Operating humidity range 20% to 80%, non-condensing 20% to 80%, non-condensing
HPLC Pump Systems
• Binary pump
- Utilizes two pump blocks and two
solvent inlet lines, enabling high-
pressure mixing of two individual
solvents. Best used for high-
throughput, high-resolution HPLC,
UHPLC, or LC-MS applications.

• Quaternary pump
- Uses a single pump block and four
solvent inlet lines to mix up to four
different solvents. These pumps offer
the broadest possible application range
with maximum flexibility in mobile
phase composition.

• Isocratic pump
- Uses a single pump block and a single
solvent inlet line. Ideal for QA/QC
analysis with refractive index detection.
HPLC Pump System’s Pump Head
 Measures of Central Tendency
❖Measure of central tendency is the representation of various values of the given data set.
❖Most important three measures of central tendency are:
‣ Mean (x̅ or μ)
‣ Median(M)
‣ Mode(Z)

❖What is Mean?
• Mean is the sum of all the values in the data set divided by the number of values in the data set. It is also
called the Arithmetic Average. Mean is denoted as x̅.
x̅ = (x1 + x2 + x3 + …… + xn) / n

❖What is Median?
• A Median is a middle value for sorted data. The sorting of the data can be done either in ascending order or
descending order. A median divides the data into two equal halves. The formula for the median is:
• If the number of values (n value) in the data set is odd, then the formula to calculate the median is,
Median = [(n + 1)/2]th term (the median is the middle data point in the list)
• If the number of values (n value) in the data set is even, then the formula to calculate the median is:
Median = [(n/2)th term + {(n/2) + 1}th term] / 2 (the median is the average of the two middle data points
in the list)
 Measures of Central Tendency
❖What is Mode?
• A mode is the most frequent value or item of the data set.
• A data set can generally have one or more than one mode value. If the data set has one mode then it is
called “Uni-modal”. Similarly, If the data set contains 2 modes then it is called “Bimodal” and if the data
set contains 3 modes then it is known as “Trimodal”.
• There is no mode for a data set if every number appears only once.

Mode = Highest Frequency Term

❖Range
• It is the difference between the highest value and the lowest value. It is a way to understand how the
numbers are spread in a data set
 Terms used for precision of results
❖Standard Deviation
• The standard deviation is a measure of how precise the average is, that is, how well the individual numbers
agree with each other.
• It is a measure of a type of error called random error - the kind of error people can’t control very well.
• It is calculated as follows:

❖Relative Standard Deviation

• Relative standard deviation is a type of standard deviation. It tells us whether the value of standard deviation
is large or small when compared with the mean value.
• The relative standard deviation (RSD) is often times more convenient.
• It is expressed in percent.
• Conceptually, it is the variability of a data set expressed as a percentage relative to its location.
• It is also known as the coefficient of variation.
• It is calculated as follows:
%RSD = [S/ x̅ ] x 100
Example for Average, SD and RSD
 Relationships between Variables

❖There are many types of relationships between two or more variables. Some of the types of relationships
between two variables are:
‣ Linear
‣ Logarithmic
‣ Polynomial

Linear Relationship Log Relationship Polynomial Relationship

 Linear Relationship
•We use regression analysis in studying linear relationships.
•Mathematically, a linear equation looks like the following:

Y = a + bx

•The two variables of interest in this equation are x and y.

•In this equation, x is called the independent variable and y is called the dependent variable.
•Using linear regression, we can find the line that best “fits” our data. This line is known as the least squares
regression line and it can be used to help us understand the relationships between the two variables.
It works by making the total of the square of the errors as small as possible (that is why it is called "least
squares"):
Correlation Coefficient of a Linear Relationship

• The correlation coefficient is a measure

for linear interdependence of the set of
data.

• It is defined between the numbers -1 and

+1.

• A correlation of value -1.0 means a

perfect negative correlation, while a
correlation of +1.0 means a perfect
positive correlation.

• A correlation of 0.0 means no linear

relationship between the movement of
the two variables.
Formula for Least Square Method Formula
Formula for Least Square Method Formula
Formula for Least Square Method Formula
Formula for Least Square Method Formula
Formula for Correlation Coefficient
 Different Kinds of HPLC Detectors
Detection Detection Destructive
Analyte requirements Principle
method limit detector?

Absorbs UV-Vis light

UV-Vis (UVD) Nanograms No Based on UV absorbance
between 190 – 800 nm
Fluorescence Has a fluorophore or labelled
Femtograms No Based on FL absorbance
(FLD) with a fluorescent tag
Based on difference between the refractive indices of
the pure mobile phase and the mobile phase
containing the analyte. Refractive index detectors are
universal detectors, requiring only that the analyte be
Refractive Index soluble in the mobile phase with a refractive index
No analyte restrictions Micrograms No
(RID) different from the solvent. When only solvent is
passing through the sample component the
measured refractive index of both components is the
same, but when an analyte passes through the flow
cell the two measured refractive index are different.
Universal detector. Able to detect all compound
which are less volatile than the mobile phase. ELSDs
works by nebulizing the column's effluents into a fine
aerosol mist, which then passes through a heated
Evaporative drift tube, where the solvent evaporates. The
Non- and semi-volatile
Light Scattering Nanograms Yes remaining non-volatile analyte particles are carried
analytes
(ELSD) further by a carrier gas to a light scattering cell, where
a beam of light illuminates them and they scatter it.
The scattered light proceeds to a photodiode which
converts it to a signal, which is proportional to the
mass of the analyte particles.
 Different Kinds of HPLC Detectors

Detection Detection Destructive

Analyte requirements Principle
method limit detector?

Measure the amount of chemicals in a sample by

creating charged aerosol particles which are
detected using an electrometer. It is commonly
Charged Aerosol Non- and semi-volatile
Picograms Yes used for the analysis of compounds that cannot be
(CAD) analytes
detected using traditional UV/Vis approaches due
to their lack of a chromophore. Signal is
proportional to the mass of the analyte particles.
Mass
Volatile and semi-volatile
Spectrometry Picograms Yes Measure the mass-to-charge ratio of ions.
ionizable analytes
(MS)
Different Kinds of Waves
 Working of a HPLC UV Detector- VWD
• For UV-Vis detection, there are three types of detectors: a variable wavelength detector (VWD), diode array
detector (DAD), or multiple wavelength detector (MWD).

• Invariable wavelength detection, a single chosen wavelength from the UV-Vis spectrum illuminates the
sample.

• In contrast, diode array and multiple wavelength detectors exposes the sample to the entire spectrum
instead of a single chosen wavelength.

Illustration of UV-Vis detection mechanism with a VWD

 Working of a HPLC UV Detector- DAD
• Diode array and multiple wavelength detectors both use a grating to disperse the light onto a photodiode
array after the light has passed through the flow cell. As a result, the absorption of all wavelengths is
simultaneous, giving the analyte a full absorption spectrum.

• This detection method is preferred when analyzing complex mixtures or samples of unknown composition,
for example, during method development or peak purity analysis.

Illustration of UV-Vis detection mechanism with a DAD

UV Absorption of Common Solvents and UV Cut Off

• Every solvent has its specific absorbance

cutoff wavelength. Below this wavelength the
solvent itself absorbs the light.

• When choosing a solvent be aware of its cutoff

and where your desired analytes will absorb. If
the wavelengths are close, choose a different
solvent.

• UV cut off is defined as the wavelength at

which the pure component has an absorbance
of 1 Absorbance Unit (AU) in a 1-cm path-
length cell with water as reference.
UV Absorption Spectrum of Common Solvents
UV Absorption Spectrum of Caffeine

The UV absorption
spectrum of
caffeine exhibits a
pair of absorption
bands
peaking at 205 nm
and 273 nm with a
characteristic
absorption
shoulder between
them.
 HPLC Calibration

HPLC Calibration

A. Pump B. Auto-sampler C. Column Oven D. Detector

Calibration/Solvent Calibration
delivery System

1. Flow Rate 1. Injection Volume

1. Detector Linearity
Accuracy (Isocratic) Accuracy

2. ComposiGonal
2. Injection Volume 2. Wavelength
Accuracy
Precision Accuracy
(Gradient Profile)

3. Flow Rate 3. Injection Volume

Consistency Linearity

4. Auto-sampler
Temperature
Calibration