HIGH PERFORMANCE

LIQUID
CHROMATOGRAPHY
RAKSHA C.A
1ST SEM ,M PHARM
DEPT OF PHARMACEUTICS
MSRUAS
CONTENTS

• Parameters used in HPLC

• Derivatisation and its types
• Applications
• References

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Parameters

1. Retention time
2. Retention volume
3. Retention factor
4. Selectivity
5. Resolution
6. Height Equivalent to a Theoretical Plate (HETP)
7. Efficiency
8. Asymmetry factors
9. Other factors
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M

Retention time
The time between the injection of a sample to the appearance of the peak at
maximum height at the detector. Each compound has a characteristic retention
time on a specific column under a given set of conditions. Therefore this
information is used to qualitatively identify the compounds in the sample.

 tm dead time

ts Time duration of analyte retained in the
stationary phase

Dead time-it is the time taken for the mobile phase to pass through the column from injector to detector 4
Retention volume (Vr)
It is the volume of mobile phase that is required to elute a substance from a column.
Retention volume = Retention time × flow rate
Selectivity (α)
It is a measure of the time or distance between the maxima of two peaks. It the ratio in capacity
factors. Selectivity can be changed by changing the mobile phase constituents or changing the
stationary phase.

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 Resolution
It is an essential HPLC performance indicator that measures how quickly and thoroughly target
components in a sample separate as they pass through a column. The difference between peak
retention times and average peak width are divided to determine resolution.
If two peaks have peak widths W1 and W2 and the respective retention times are t1, and t2 then
the resolution (R) of the two peaks is given

When two components in a sample are poorly resolved the peaks overlap to an extent that
makes identification and determination of the components impossible.
Resolution can be improved by
(i) changing the temperature
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 Retention Factor
Retention Factor (k) Formerly referred to as capacity factor or k prime
It measures the period of time that the sample component resides in a
stationary phase relative to the time it resides in the mobile phase. It is
calculated from the retention time divided by the time for an unretained peak
(t0).
When k is < 1.0, separation is poor
When k is > 30, separation is slow
When k is = 2-10, separation is good

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Height Equivalent to a Theoretical Plate (HETP)
A theoretical plate is an imaginary or hypothetical unit of a column where distribution of
solute between stationary phase and mobile phase has attained equilibrium. It can also be
called as a functional unit of the column. It can be of any height, which describes the
efficiency of separation. If HETP is less, the column is more efficient and and If HETP is more,
the column is less efficient.
HETP = length of the column/ no. of theoretical plates
HETP is given by Van deemter equation
HETP = A+B/u+Cu
 A = Eddy diffusion term or multiple path diffusion which arises due to the packing of the
column. This can be minimized by uniformity of packing.
 B = Longitudinal diffusion term or molecular diffusion.
 C = Effect of mass transfer.
 u = flow rate or velocity of the mobile phase. 9
 Efficiency
• Efficiency of a column is expressed by the theoretical plates(N).
If No of theoretical plates Is high, the column is said to be highly efficient.
For GLC, a value of 600/metre is sufficient. But in HPLC, high values like
40,000 to 70,000/ meter are recommended.

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 Asymmetry
A peak is considered asymmetric when the distance from the start of the peak
to the centre (A) and from centre to the end (B) of the peak differs. Within
asymmetric peaks, there are two possibilities that could exist;
• Fronting and
• Tailing
If a peak has a wider front half (distance A) compared to the back half (distance
B), it called “fronting” (or “leading”) and vice versa is known as “tailing”

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• Fronting is due to saturation of stationary phase and can be avoided by using less
quantity of sample.
• Tailing is due to more active adsorption sites and can be eliminated by support
pretreatment. Asymmetry factor can be calculated by ;
ASF = b/a
a - represents the width of the front half of the peak,
b - is the width of the back half of the peak.
The values are measured at 10 % of the peak height from the leading or trailing
edge of the peak to a line dropped perpendicularly from the peak apex .
For T > 1 the peak profile is named tailing.
For T < 1 the peak profile is named fronting.
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Other common PARAMETERS to be considered include
• internal diameter
• particle size
• pore size
• pump pressure.
For different compounds the parameters can be changed according to
their nature and chemical properties.

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Internal diameter and particle size
Internal diameter: it is a critical aspect that determines quantity of analyte that
can be loaded onto the column and also influences sensitivity. Larger columns are
usually used in industrial applications. Low ID columns have improved
sensitivity and lower solvent consumption at the expense of loading capacity.
3.9 to 4.6 mm and 15, 25, and 30 cm in length
Particle size: Most traditional HPLC is performed with the stationary phase
attached to the outside of small spherical silica particles (very small beads).
Smaller particles generally provide more surface area and better separations
3-5 μm

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 Pore size and pump pressure
Pore size: Pore size defines an ability of the analyte molecules to penetrate
inside the particle and interact with its inner surface. Many stationary phases
are porous to provide greater surface area.
Small pores provide greater surface area while larger pore size has better
kinetics especially for larger analytes. It is especially important because the
ratio of the outer particle surface to its inner one is about 1:1000. The surface
molecular interaction mainly occurs on the inner particle surface.
Pump pressure: Pumps vary in pressure capacity, but their performance is
measured on their ability to yield a consistent and reproducible flow rate.
Modern HPLC systems works at much higher pressures, and therefore be able
to use much smaller particle sizes in the columns (< 2 micrometres).
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 Derivatization
Derivatization is a technique that converts a compound into products of similar
chemical structure, a derivative.
In general analytical derivatization is for the below main reasons :
permit analysis of compounds with inadequate volatility (or) stability.

is for improve chromatographic behaviour or detectability.

reduce tailing factor and in the improvement of resolution for the compounds which contains -OH, -COOH, =NH and -NH2
functional groups.

required for Sensitivity enhancement and selectivity alter retention characteristics, and provide selective response for analytes
in complex matrices.
•.

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 Derivatization reagent
• They are added to chemically modify a compound to produce a new product or
compound for easy analysis in LC, HPLC and GC.
.
Characteristics
• The reagent should produce the derivatives, which shoudn’t react with column.
• The derivatizing reagents should produce the derivatives with in the short time.
• Derivatizing agent should not cause the loss of the sample during the reaction
• Reagent should not cause the structural alternatives during the process of
derivatization
• The reagent should have the potency to produce 95% of derivatives.
• ex -If functional group is Alcohols /Phenols/ carboxylic acid/ amines /amides,then
derivatization reagent is Dimethylformamide
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 CLASSIFICATIONS
• The derivatization reactions are categorized based on selection of reagent and type of
substitution reaction:
• Alkylation • Silylation • Acylation
• Alkylation- The procedure involves protection of active hydrogens in a sample molecule
by the replacement of active hydrogen with aliphatic or aliphatic- aromatic group by
esterification process. Ex Benzyl group.
• Silylation it is the most commonly used method for the derivatization of non-volatile
chemical compounds into volatized samples. It was the best method suitable for the
volatile sample’s analysis in chromatography. This method is the introduction of a silyl
group into a molecule. Ex- Trimethyl chrorosilane .
• Acylation - Thioesters, amides and esters are generated by acylation of active hydrogens
(e.g., -OH, -SH and -NH). It is also popular method for the production of volatile
derivatives and also generating highly polar and in volatile organic materials.19 Stable
There are 2 types of derivatizations
1. pre column derivatization and
2. post column derivatization
pre column derivatization
• Performed before the analytical separation is attained.
• Sample is derivatized manually or automatically and injected into the HPLC
column.
• Separation of components occurs after derivatization
• techniques are mostly used for the analysis of amino acids likeo-
phthalaldehyde, phenyl isothiocyanate (PITC), fluoresceine and dansyl
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POST COLUMN DERIVATIZATION
• Performed after analytical separation of compound but prior to detection.
• Addition pump is used for addition of derivatizing agent to the elute sample from column.
• O-phthalaldehyde (OPA), ninhydrin are mainly used as derivatization in reagents.
ADVANTAGES OF DERIVATIZATION
• improve the chromatographic properties of an analyte, making it easier to detect and
quantify.
• It can also improve the sensitivity of the analysis, allowing for lower detection limits and
better resolution of the peaks.
• an be automated, allowing for high-throughput analysis of large numbers of samples.

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 LIMITATIONS
• The reagents used can be expensive and difficult to obtain.
• The technique is also time-consuming, as the derivatization process must be
completed before the separation can begin.
• The technique is also limited by the type of molecule being analyzed, as some
molecules may not be amenable to derivatization.
• It can also lead to the formation of artifacts, which can interfere with the
analysis and lead to incorrect results.

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 Applications
• In quality control, it is used to check if the manufactured products comply with the specified
standards. These specific standards are fixed by the pharmacopeias and other drug
regulating bodies. The guidelines mentioned in the pharmacopeia will give an idea of how
the peak of the drug in the formulation should look when run with specified 
HPLC mobile phases are used. If the peaks do not correspond to those shown in the
pharmacopeia, the batch cannot be passed for quality check.
• It is used for bioavailability studies, drug release from the formulation, dissolution studies,
etc. After a formulation is designed, the drug release over some time is tested in
bioavailability studies. Then the sample released is taken and injected into the HPLC system
to note the individual molecules released in terms of quantity. Since the molecules might be
similar, their separation is easier over the column under pressure and detection becomes
easier as the system is connected UV-visible detector or other specified detectors.
For this, the drug formulations like injections, solutions, a dissolved form of solid dosage
forms are injected into the HPLC injector to record the peaks of the individual constituents.
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• Also, any new molecule under development or in a preclinical trial is analyzed to see
their blood concentration after certain intervals of administration. This helps to
evaluate the metabolic profile, plasma concentration, bioavailability, etc., of the
formulation or chemical moieties under development.
• In-plant constituents, there are many molecules with similarity in chemistry like
isoflavones, glycosides, saponins, etc. but they have different activity or nutritional
value. Other methods can’t precisely determine these compounds. Hence they are
determined by HPLC analysis through separation into individual components and
thereby identification
• Assay of cephalosporins ,frusemide, theophylline ,corticosteroids
dichlorphenamide can be done.

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• In separation of compounds -in HPLC, a liquid mobile phase (MP) carries a mixture of
compounds through a column packed with particles. As a pump forces the MP through
the column, the components of the mixture interact with the stationary phase - the
particle’s surface - to different degrees and are separated in the process.
• Identification- in R & D, it is used to identify the specific molecule or component in the
mixture under research by comparing each peak’s retention time [t R] with that of
injected reference standards in the same chromatographic system [same mobile and
stationary phase], to determine the purity of the substance.
• Stability - due to its high-resolution capacity, sensitivity and specificity. Non-volatile,
thermally unstable compounds can also be analyzed by this technique.
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ISOLATION OF NATURAL PHARMACEUTICALLY ACTIVE COMPOUNDS
Some plant alkaloids and glycosides can be isolated as stated below :

• In forensic studies
these machines help forensic scientists analyze volatile substances such as gunpowder residue, fibers,
and toxins. One of its most common uses is to determine materials used in explosives. Additionally, many
explosive materials can wear down during the gas chromatography methods. Because a high-pressure pump is
moving the substance through liquid instead of gas, it keeps the substance in its most pure state to ensure
accuracy.

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 References
• Malviya, Rishabha & Bansal, Vvipin & Pal, Om & Sharma, Pramod. (2010). High performance liquid chromatography:
A short review. Journal of Global Pharma Technology. 2. 22-26.
• Ranga.nr, Peters, C. and Himanshu (2021) 5 application of HPLC: In diagnosis, Research & Pharma Industry, Study
Read. Available at: https://www.studyread.com/application-hplc-pharmaceutical-industry/ (Accessed: January 30,
2023).
• CH Prudhviraju., et al. “Pre and Post Column Derivatization of Amino Acid - A Systematic Review of HPLC". Acta
Scientific Pharmaceutical Sciences 5.8 (2020): 104-105.
• Sankar, Ravi (2020). Current Trends in Performance of Forced Degradation Studies and Stability Indicating Studies of
Drugs. 10.9790/3008-1206021736. 21
• Factors affecting resolution in HPLC. Available at:
https://www.sigmaaldrich.com/IN/en/technical-documents/technical-article/analytical-chemistry/small-molecule-
hplc/factors-affecting-resolution-in-hplc (Accessed: January 30, 2023).
• shankar, ravi (2019) Fundamental chromatographic parameters - researchgate. International Journal of
Pharmaceutical Sciences Review and Research · . Available at: https://www.researchgate.net/profile/Ravi-Sankar-
24/publication/340849229_Fundamental_Chromatographic_Parameters/links/5ea0882392851c87d1acd72c/
Fundamental-Chromatographic-Parameters.pdf?origin=publication_detail (Accessed: February 5, 2023).
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…Thank you…

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