Introduction to HPLC
High Performance Liquid Chromatography (HPLC) is one of the most widely used techniques in modern laboratories. It is essential in many sectors, from pharmaceutical research and NHS diagnostics to environmental monitoring, food safety testing, and forensic science.
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HPLC is a powerful technique for separating, identifying, and measuring chemicals in a mixture. If you work in laboratory science, there’s a very good chance you will use HPLC at some point in your career. ​In fact, employers often list HPLC knowledge and experience as one of the top skills they look for when hiring laboratory staff.
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At first, the theory and equipment can seem complicated — but don’t worry we’ll take a step-by-step approach to understanding how HPLC works. You’ll get hands-on experience with an HPLC simulator, analyse real data, and prepare professional scientific reports.
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Introduction to HPLC
On the face of it, paper chromatography and HPLC seem worlds apart, but actually when it comes to the separation of mixtures of compounds, the theory and principle of separation are essentially the same. ​Both techniques require a stationary phase (SP) and a mobile phase (MP).
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For paper chromatography the SP is filter paper (a porous material of intertwined cellulose fibres) and the MP is typically water. As the MP travels up the SP (by capillary attraction) the compounds partition between the two phases at different rates dependent on their chemical structure and properties.
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If a compound is highly soluble in water and only weakly binds to the SP it will move quickly up the paper.
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If a compound is poorly soluble in water and strongly binds to the SP it will move slowly.
The Retention factor (Rf) is used as a measure of degree of separation compared to the solvent front.
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In this example the SP is spotted with a black dye and a known red dye, for reference. We can observe that the black dye is made of a mixture of blue, green and red dyes - and the red dye has the same Rf value as the reference spot which suggest that they are the same compound.
Click to play
Water
Retention factor Rf =
distance of spot (cm)
solvent front (cm)
Introduction to HPLC
For HPLC, the stationary phase (SP) is silica.
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Silica is a very fine powder resin (fig 1), that can be sorted and refined to obtain the required particle size - fig 2 shows a zoomed in image of the spherical porous particles.
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​To make an HPLC column, the manufacturers pack steel columns (fig 3a) with a slurry of the silica, under very high pressure, Fig 3b shows an enlarged image of the column with the inlet fitting removed showing the compacted silica that looks like a solid white rod.
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​The separation of mixtures of compounds using an HPLC column works in the same way as paper chromatography. As the compound mixture is pushed through the column in the mobile phase (MP), each compound binds to the stationary phase (SP) at different rates and the compounds exit the column as separate peaks (fig 4). Instead of Rf values we measure the retention time (RT) of each peak, which is the time it takes from the point of injection of the sample (to) to the time the compound passes through the detector UV cell, with (tend) recorded at the peak apex.
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RT = tend - to (measured in minutes or seconds)
Fig 1: silica resin
Fig 2: silica particles
Fig 3a: HPLC column 50mm x 2.1mm
Fig 3b: inlet removed to show the compacted silica resin
Fig 4: separation of a black dye on an HPLC column
Click inside the column to play
Reverse phase HPLC
Fig 4
What do we mean by 'reverse phase' HPLC?
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The original application of silica in chromatography involved the use of bare non derivatised silica (fig 4) that is formed of long branched chains of Si-O-Si bonds with highly polar hydroxyl groups. Compounds are separated using non-polar mobile phases, e.g. mixture of 97% hexane and 3% isopropanol. Compounds bind, via 'hydrogen bonding' interactions to the Si-OH groups, consequently non polar compounds are weakly retained and elute early and polar compounds bind strongly resulting in long RT's. This type of chromatography is called Normal Phase.
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Reverse phase chromatography involves the use of derivatised silica whereby the hydroxyl groups are reacted to attach a long alkyl chain, e.g. C8 (fig 5) which has 7 methylene (-CH2) groups and a methyl (-CH3) group - this dramatically changes the property of the silica particles to form a non polar surface. And compounds are separated using polar mobile phases e.g. mixture of 65% water and 35% methanol.
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Fig 5
Since bare silica and non polar solvents were in use long before the introduction of derivatised silica, it was retrospectively named 'normal phase'.
Derivatised silica columns (e.g. C8), using polar mobile phase solvents (i.e. the opposite of normal phase) where subsequently named as 'reverse phase' columns.
Both techniques are used in research and industry, but reverse phase has a far wider range of applications and it will be the focus for the rest of the training course.​
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There are a range of commercial reverse columns available, but the most frequently used is the C18 column.
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Reverse phase HPLC
What is the mechanism that controls the separation of compounds by reverse phase HPLC?
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At the molecular level, the C18 carbon chains (attached to the silica particles) are immiscible with the mobile phase solvents - we can think of them as two separate layers. When a sample (containing different compounds) enters the column, the compounds can bind to the column (stationary phase) or remain in the mobile phase - the partition between the two phases and the time spent in each phase is greatly impacted by the ratio of organic to aqueous solvents - what does that mean in practice?
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Example: lets consider 3 compounds A, B and C that we want to separate on a C18 column using a mobile phase of methanol and water.
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When using a MP of 90% of methanol : 10% water.
At this high % of organic solvent, the solubility of
compounds A, B + C is high and they
bind weakly with the C18 chains. Hence all 3 compounds
pass quickly through the column (short RT's)
with little or no separation.
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When the % of organic solvent is reduced (to say 60%) the
compounds are less soluble in this mix and binding to
the C18 chains becomes energetically more favourable.
Hence the compounds spend more time bound to the
SP resulting in longer RT's. But, in this example,​ the first 2 peaks
are still co-eluting .i.e. they overlap each other.
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Lowering the % of organic solvent further (to say 40%)
results in stronger binding of the compounds to the C18
chain and longer RT's. Now we can see baseline separation
of all 3 peaks i.e. no co-eluting.
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​IMPORTANT: If all 3 compounds have identical polarities (same solubility properties) and binding affinity
to the SP we would never be able to separate the compounds no matter what % of organic we used !
Fortunately most compounds do have different polarities and exhibit different binding affinities to the SP and,
in general, we can achieve separation by changing the ratio of organic to aqueous in the MP.​​​
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When developing a new HPLC method follow this general procedure:
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Start at a high % organic in the MP to ensure that all of the compounds are eluted from the column
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Followed by step wise reduction in % organic until baseline separation is achieved.
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IMPORTANT: One of the most crucial requirements for a validated HPLC method is a stable and highly reproducible retention time (RT) value for each compound peak - ideally less than 0.5% standard deviation. When the same samples are repeatedly analysed, the measured RT for each peak needs to nigh identical. If poor reproductivity of RT's was observed it would be impossible to confidently confirm the identity of each peak (compared to RT's measured for calibration standards) and to compare data between different samples. ​​​
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Introduction to HPLC
Let's take a look at the different modules in an HPLC system.​​
Mobile phase solvents housed in a spill tray
Bottle A: aqueous solvent
Bottle B: organic solvents (e.g MeOH)
Binary Pump:
High pressure pump that draws solvents A and B and mixes at the required ratios.
Flow rate - 1 to 5 ml/min, maintaining constant flow against the high back-pressure generated by the column
Autosampler:
Draws aliquot (1 to 20 µl) from sample vial to inject onto the column.
Column oven:
Maintains column at a fixed temperature.
Range 10 to 60 C
o
UV detector:
wavelength range 190 to 600 nm
Fluidics: how the modules are connected & the flow path
WASTE
Click below here
UV cell
in
Sensor
Light
Optics slit
UV cell
out
Chromatogram
Why do we need a UV detector?
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When performing paper chromatography experiments it is highly likely that you used coloured dyes that are visible by eye.
However, the vast majority of chemical compounds are colourless, hence we need a detector to detect and measure peaks as they elute from the column.
There are a few different options available, of which the UV detector is by far the most commonly used technique for HPLC.
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How does it work?
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A UV light source passes through the transparent UV cell and is measured at the sensor. When there is only mobile phase flowing through the UV cell (i.e. before a sample is injected) the UV detector will take a 'baseline' reading - this is shown as the flat baseline in the chromatogram.
​​When a sample is injected onto the column and a compound starts to elute and flows through the UV cell, some of the UV light is absorbed by the compound causing a decrease in the amount of light measured at the sensor - this is shown as a peak on the chromatogram. The size of the peak (measure of UV absorbance) is proportional to the concentration of the compound in the UV cell (look up the meaning of the Beer-Lambert Law) and the apex of the peak is recorded as the RT for that compound.
Can all compounds be detected using a UV detector?
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Short answer, almost all compounds (approx. > 90%) can be analysed using a UV detector. If a compound has one or more chromophore group(s) that absorbs UV light (in the range 190 to 400nm) it will give a UV peak response. Chromophores are unsaturated groups containing pi bonds that can absorb UV light, examples: aromatic compounds, carbonyl groups (esters/amides/carboxylic acids/ketones etc), double (C = C) and
triple (C ≡ C) carbon bonds. Any organic compound that contains just single (C - C) bonds and simple inorganic salts will not absorb UV light and hence cannot be detected using a UV detector.
Q2. Explain the difference between paper chromatography and HPLC, including differences in stationary & mobile phases and Rf vs RT measurements.
Q1. What does the abbreviation HPLC stand for?
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Q3. What is the difference between normal phase and reverse phase HPLC?
Using an C18 column as an example discuss differences in chemical structure, surface polarity of SP and the polarity of the MP's used. How many methylene (-CH2) groups and methyl (-CH3) groups are there in each C18 chain?
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Q4. Explain why reproducible RT measurements are an important requirement for a successful, validated HPLC method.
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HPLC method development:
Product A, an energy drink currently on the market, undergoes HPLC quality control checks at the manufacturing plant using method LC-25A based on:
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Reverse phase column : C18
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Mobile phase: 60% A (water) and 40% B (methanol).
Using this method baseline separation is achieved for all 3 active ingredients, ensuring accurate quantitative QC analysis.
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The manufacturer is developing a new Product B which contains the same 3 active ingredients as A, plus a 4th new ingredient. Initial analysis of B using the existing HPLC method LC-25A show that the peak for the new ingredient is co-eluting with one of the other peaks.
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Q5. Explain what is meant by the terms 'baseline' separation and 'co-eluting' peaks. What changes to the method would you investigate to solve the problem ? - explain your reasoning.
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Q6. Select a module (in the left stack) to build an HPLC system that requires the minimum length of tubing connecting each module.
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Q7. Why do we need a high pressure pump for HPLC?
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Q8. Why is the HPLC column housed inside an oven?
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Q9 Describe the function of the autosampler, including the steps performed to inject an aliquot of sample onto the column.
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Q10. Explain how the UV detector is used to detect compounds that elute from the HPLC column. Include a short discussion about the chemical structural features required to generate a UV signal.
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HPLC analysis of OTC product Anadin Extra
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Let's apply the theory by analysing samples and evaluating real HPLC data. ​ Your course leader will provide you with a full lesson plan for the quantitative analysis of Anadin Extra, an 'over the counter' (OTC) pain relieve tablet product.
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During this module we'll cover the following;
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1. HPLC method development:
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Separation of aspirin, paracetamol and caffeine
2. Peak identification
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Analysis of QC samples
3. Manual and automated Sequence analysis
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Calibration
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Quantitative analysis
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Data analytics
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Before starting the experiment, calculate the concentrations of the calibration and quality control standards.
Preparation of calibration standards:
Stock solution (labelled STD4) was prepared by weighing Paracetamol (0.2296g), Aspirin (0.3576g) and Caffeine (0.0526g) into separate weigh boats and transferring each to a 100ml volumetric flask - each boat was washed with approx 10ml of methanol into the flask. Approx 50ml of methanol was added and the solution vigorously shaken to ensure that all 3 compounds were fully dissolved. Finally, the volumetric flask was made up to the mark with methanol and gently shaken and capped.
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The other standards were prepared by pipetting an aliquot of STD 4 into a 5ml volumetric flask and making to the mark with methanol. Approx 1ml of each were pipetted into HPLC vials with a septum piercing caps.
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Standard Volume of STD 4 (ml) Vial number
​STD1 3.50 51
STD2 4.00 52
STD3 4.50 53
STD4 - 54
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Calculate the concentration of each compound, mg/ml to 3 decimal places - input the values in the table below.
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Vial :
ID
Paracetamol mg/ml
Aspirin mg/ml
Caffeine mg/ml
51 STD1
Try again
?
score:
Preparation of quality control standard QC 1:
Paracetamol (0.0992g) was weighed directly into a 50ml volumetric flask and made to the mark with methanol. Vial 71.
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Preparation of quality control standard QC 2:
Aspirin (0.1486g) was weighed directly into a 50ml volumetric flask and made to the mark with methanol. Vial 72.
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Preparation of quality control standard QC 3:
Caffeine (0.0224g) was weighed directly into a 50ml volumetric flask and made to the mark with methanol. Vial 73.
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Calculated the concentration of each QC sample, mg/ml to 3 decimal places - input the values in the table below.
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Vial :
ID
Paracetamol mg/ml
Aspirin mg/ml
Caffeine mg/ml
71 QC1
Try again
?
score:
DAD
Ready
215
nm
Column oven
Ready
C18: 5um : 50 x 4.6mm
40
o
C
Pump
Ready
25%
75%
A
B
Flow rate
2.00
ml/min
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Autosampler
Ready
Vial number
5.0 ul
inject volume
41
Mismatch -check vial and method
selection
Autosampler webcam
Sequence file ID | Vial | Sample Name | Sample Type c/q/s |
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Sequence file ID | Vial | Sample Name | Sample Type c/q/s |
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Sequence file ID | Vial | Sample Name | Sample Type |
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