High-Performance Liquid Chromatography (HPLC)

High-pressure liquid chromatography, now known as high-performance liquid chromatography

(HPLC), is a chromatographic technique used to identify, quantify, separate and purify
individual compounds present in a mixture [1].

How does high-performance liquid chromatography work?

In high-performance liquid chromatography (HPLC) the sample mixture is passed along with a
liquid solvent under high pressure through a column filled with a solid adsorbent material. The
pressure within the system is built-up with the help of pumps. The working principle is that each
compound in the mixture interacts slightly different with the adsorbent material in the column,
resulting in varying flow rates for the different components. This leads to separation of the
components as they flow out of the column. The adsorbent material used is typically granular,
made up of solid particles, such as silica, constituting the ‘stationary phase’. The pressurized
liquid is a mixture of solvents, such as water and organic liquids like methanol and acetonitrile,
which constitute the ‘mobile phase’.

The detector is connected to a digital microprocessor and user software for data acquisition and
analysis. The separated compounds are visualized as peaks with the number of peaks
corresponding to the number of separated components in the mixture. The area of the peak is
proportional to the concentration of the compound present within the mixture [2]. The resolution
between two peaks in chromatographic techniques is the extent to which substances are
separated during the experiment. Higher resolution reflects good separation of the compounds.

What is high-performance liquid chromatography used for?

High-performance liquid chromatography has a wide range of applications in the fields of

biochemistry, analytical chemistry, pharmaceuticals, forensics, food research, and other areas.

Why would you use high-performance liquid chromatography?

HPLC is both affordable and adaptable. It provides useful benefits such as data management and
instrument validation.
 High Performance Liquid Chromatographic Columns

High performance liquid chromatography (HPLC) is a type of liquid chromatography that uses a
liquid moblie phase. The same basic principals from gas chromatography are applied to liquid
chromatography. There are three basic types of liquid chromatographic columns: liquid-liquid,
liquid-solid, and ion-exchange. Liquid-liquid chromatographic columns have the liquid
stationary phase bonded or absorbed to the surface of the column, or packed material. liquid-
liquid chromatographic columns are not as popular because they have limited stability and they
are inconvenient. Partitioning occurs between the two different liquids of the mobile and
stationary phases. In liquid-solid chromatographic columns the stationary phase is a solid and the
analyte absorbs onto the stationary phase which separates the components of the mixture. In ion-
exchange chromatographic columns the stationary phase is an ion-exchange resin and
partitioning occurs with ion exchanges that occur between the analyte and stationary phase.

Usually HPLC has a guard column ahead of the analytical column to protect and extend the life
of the analytical column. The guard column removes particulate matter, contaminants, and
molecules that bind irreversibly to the column. The guard column has a stationary phase similar
to the analytical column.

The most common HPLC columns are made from stainless steel, but they can be also made out
of thick glass, polymers such as polyetherethelketone, a combination of stainless steel and glass,
or a combination of stainless steel and polymers. Typical HPLC analytical columns are between
3 and 25 cm long and have a diameter of 1 to 5 mm. The columns are usually straight unlike GC
columns. Particles that pack the columns have a typical diameter between 3 to 5 µm. Liquid
chromatographic columns will increase in efficiency when the diameter of the packed particles
inside the column decreases.

Packing Material

HPLC columns are usually packed with pellicular, or porous particles. Pellicular particles are
made from polymer, or glass beads. Pellicular particles are surrounded by a thin uniform layer of
silica, polystyrene-divinyl-benzene synthetic resin, alumina, or other type of ion-exchange resin.
The diameter of the pellicular beads is between 30 and 40 µm. Porous particles are more
commonly used and have diameters between 3 to 10 µm. Porous particles are made up silica,
polystyrene-divinyl-benzene synthetic resin, alumina, or other type of ion-exchange resin. Silica
is the most common type of porous particle packing material.

Partition HPLC uses liquid bonded phase columns, where the liquid stationary phase is
chemically bonded to the packing material. The packing material is usually hydrolyzed silica
which reacts with the bond-phase coating. Common bond phase coatings are siloxanes.

1. Reverse and Normal Phase HPLC

A polar stationary phase and a non-polar mobile phase are used for normal phase HPLC. In
normal phase, the most common R groups attached to the siloxane are: diol, amino, cyano,
inorganic oxides, and dimethylamino. Normal phase is also a form of liquid-solid
chromatography. The most non-polar compounds will elute first when doing normal phase
HPLC.

Figure 11: Basic structure of a siloxane. The R groups can be

varied depending on the type of column and analyte being analyzed. This figure was created with
ChemBioDraw Ultra 12.0.

Reverse phase HPLC uses a polar mobile phase and a non-polar stationary phase. Reverse phase
HPLC is the most common liquid chromatography method used. The R groups usually attached
to the siloxane for reverse phase HPLC are: C8, C18,or any hydrocarbon. Reverse phase can also
use water as the mobile phase, which is advantageous because water is cheap, nontoxic, and
invisible in the UV region. The most polar compounds will elute first when performing reverse
phase HPLC. Check the animation on the principle of reversed-phase chromatography to
understand its principle.

2. Ion Exchange Chromatographic Columns

Ion exchange columns are used to separate ions and molecules that can be easily ionized.
Separation of the ions depends on the ion's affinity for the stationary phase, which creates an ion
exchange system. The electrostatic interactions between the analytes, moble phase, and the
stationary phase, contribute to the separation of ions in the sample. Only positively or negatively
charged complexes can interact with their respective cation or anion exchangers. Common
packing materials for ion exchange columns are amines, sulfonic acid, diatomaceous earth,
styrene-divinylbenzene, and cross-linked polystyrene resins. Some of the first ion exchangers
used were inorganic and made from aluminosilicates (zeolites). Although aluminosilicates are
not widely used as ion exchange resins used.

3. Size Exclusion Chromatographic Columns

Size Exclusion Chromatographic columns separate molecules based upon their size, not
molecular weight. A common packing material for these columns is molecular sieves. Zeolites
are a common molecular sieve that is used. The molecular sieves have pores that small molecules
can go into, but large molecules cannot. This allows the larger molecules to pass through the
column faster than the smaller ones. Other packing materials for size exclusion chromatographic
columns are polysaccharides and other polymers, and silica. The pore size for size exclusion
separations varies between 4 and 200 nm.
 Figure 22: Schematic of a size exclusion column. The
larger particles will elute first because they are too big to fit inside the pores. The smallest
particles will elute last because they fit very well inside the pores. This figure was created with
Microsoft Paint.

4. Chiral Columns

Chiral columns are used to separate enantiomers. Separation of chiral molecules is based upon
steriochemistry. These columns have a stationary phase that selectively interacts with one
enantiomer over the other. These types of columns are very useful for separating racemic
mixtures.

Column Efficiency

Peak or band broadening causes the column to be less efficient. The ideal situation would to have
sharp peaks that are resolved. The longer a substance stays in the column it will cause the peaks
to widen. Lengthening the column is a way to improve the separation of different species in the
column. A column usually needs to remain at a constant temperature to remain efficient. Plate
height and number of theoretical plates determines the efficiency of the column. Improving the
efficiency would be to increase the number of plates and decrease the plate height.

The number of plates can be determined from the equation:

N=L/H(1)(1)N=L/H
or HETP can also be determined by the van Beemter equation:

H=A+Bu+Cu(5)(5)H=A+Bu+Cu

where H equals HETP, A is the term for eddy diffusion, B is the term for longitudinal diffusion,
C is the coefficient for mass-transfer between the stationary and mobile phases, and u is the
linear velocity.

Silica based columns

General guidelines Silica is the ideal support for HPLC columns. It offers a large mechanical stability,
excellent physicochemical surface properties, a wide range of bonding chemistries and is compatible
with a broad range of organic solvents. However, the following points are extremely important to know
when working with silica based HPLC columns.

• pH stability

In general, HPLC columns are stable within a pH range of 2 to 8. If you are measuring a pH value, the
measurement must be done in the aqueous media before mixing the eluent with organic solvents.
Modern HPLC columns can be used outside that pH range. The new bonding chemistries allow use down
to pH 1 for some stationary phases. However, please check vendor’s product information before using
silica based column outside the pH range of 2 to 8. However, best lifetimes are obtained between pH 2.0
and pH 6.8. Stationary phases based on ultra pure silica gel can also be used at higher pH ranges, up to
pH 11, depending on the chemical nature of the modifier used in the mobile phase. Large bases (like
Pyrolidine) are not able to attack the surface of the silica and therefore can be used at higher pH values.
If you are working at pH values above 8 with small bases as modifier (like Ammonia), we highly
recommend using stationary phases based on Polymers or Zirconiadioxide.

• Mechanical stability

Stationary phases based on silica are mechanically very stable. The packed columns show no pressure
limit and can be used at more than 40 MPa (6000 psi) without any problem. However, please avoid
pressure shocks on the column. Pressure shocks lead to channeling in the column, which results in peak
splitting in the corresponding chromatogram.

• Mobile phases (Eluents)

Silica based stationary phases are compatible with all organic solvents in the above mentioned pH
range. Please use the highest quality solvents available (HPLC grade). Also, please filter all prepared
buffer through a 0.5 µm filter before using them in your HPLC system. Always keep in mind; your column
is the best filter! The use of non pure solvents in HPLC causes irreversible adsorption of impurities on
the column head. These impurities block adsorption sites, change the selectivity of the column and lead
to peak splitting in the chromatogram. In gradient elution, impurities cause so called “Ghost Peaks”.
Ghost peaks are peaks that always appear in the same position on the chromatogram. Their origin is not
the sample, but the impurities from the solvents or solvent additives. Therefore, it is highly
recommended to run a gradient without injection in the beginning of each method to determine the
ghost peaks. To avoid irreversible adsorption at the column head, you should always use a pre-column.
The use of a pre-column increases the life time of a column dramatically. In addition, a pre-column can
filter solid parts stemming from pump seals or injection rotors. An alternative to a pre-column is an in-
line filter. These filters are attached directly to the column. These filters get rid of solid parts in the
eluent but will not avoid irreversible adsorption of organic impurities. Proper storage of HPLC columns •
For short term storage, i.e. over night, columns

What are the different types of contaminants in water that affect HPLC
results?

1. Organics

Organic contamination of ultrapure water may affect chromatographic separation in different

ways:

(i) Reducing column life - Organic molecules that bind to the surface of the column can slow-
down access of sample and solvent molecules to the binding sites within the column beads
(stationary phase). This results in a decrease in the column’s ability to separate compounds or
loss of resolution and a shorter column life.

(ii) Reducing sensitivity - Organic molecules in the eluent water may compete with sample
molecules for binding to the column beads (stationary phase). This decreases the number of
sample molecules binding to the column, consequently reducing the number of molecules
released during the elution process.

(iii) Inaccurate data - Contaminant or ghost peaks can be obtained from organics that may
accumulate at the head of the column and later get collected as eluate.

(iv) Shift in retention time - High levels of organics can create a new stationary phase in the
column, which can cause a shift in retention time and peak tailing. It can also lead to back
pressure increase.

It is, therefore, critical to accurately monitor the level of organics in water used for HPLC
applications. Total organic carbon (TOC) is a measure of the total organic species present in
water. TOC is measured in units of parts-per-million (ppm) or parts-per-billion (ppb). Organics,
present in high ppb, can alter spectral identification of trace components within the mixture and
influence peak quantification.

An HPLC system can be contaminated by a large variety of TOC sources. These include water,
leaching from purification media, tubing and containers, bacterial contamination and potentially,
absorption from the atmosphere. In fact, it is now recommended to avoid using high-purity
bottled water that is not freshly purified. This is because HPLC bottled water that has been
standing in a laboratory environment for more than 8 h (exposure to atmospheric organics) or
distilled water is susceptible to an increase in TOC levels.
Fig 1: Comparison of the TOC levels in HPLC-grade water and Ultrapure water.

Figure 1 compares the chromatogram output from HPLC-grade water and ultrapure water
measured at wavelengths of 254 nm and 214 nm. HPLC-grade water has higher TOC levels,
which elute out from the column causing baseline shifts, with an increased size and number of
peaks, compared to ultrapure water [2].

Therefore, the use of pure water is critical for HPLC analysis, and it is important to keep water
free from all sorts of contaminants. Chromatographers, in addition to ensuring the purity of
organic solvents, standards and other HPLC mobile phase components, must also ensure that the
reagent water is of high quality and devoid of any contaminants.
2. Particles and Colloids

The presence of particles and colloids in the water sample can cause damage to the pump and
injector in addition to physically blocking the column. These can also behave as a solid phase
within the column binding to the sample constituents. Colloids can also irreversibly adsorb to the
column packing material thereby preventing the sample constituents from binding to the column.

3. Ions

The presence of ions in the solvent can also affect chromatographic separations. The presence of
any UV-absorbing ions, such as nitrates, nitrites, sulfates, bromides, chlorides, and fluorides, can
pass through the column and appear as a peak in the chromatogram making the data difficult to
analyze.

Among the different water contaminants that affect HPLC analysis, organics are by far the most
important determinants for water purity. Experimental evidence has strongly suggested that
freshly prepared ultrapure water should be the choice for any HPLC as other sources of water,
namely distilled water or even HPLC-grade bottled water still contain relatively high amounts of
organics, which can compromise the quality of the chromatograms and the performance of the
apparatus (see, Figure 1).

It is, therefore, imperative to ensure that high standard water purification systems are used and
the system itself is properly maintained.
REFERENCES

1. Skoog, D., Holler, J., Crouch, S. Principles of Instrumental Analysis, 6th

Ed.; Thomson Brooks/Cole: Belmont, 2007.
2. Poole, C.F. The Essence of Chromatography; Elsevier: San Francisco,
2003.
3. Miller, J.J. Chromatography: Concepts and Contrasts, 2nd Ed.; John Wiley
& Sons, Inc.: Hoboken, NJ, 2005.