UNIT
UNIT 8
8 HIGH
HIGH PERFORMANCE
PERFORMANCE LIQUID
LIQUID
CHROMATOGRAPHY (HPLC)
CHROMATOGRAPHY (HPLC)
Structure Structure 8.1 8.1 IntroductionIntroduction Objectives Objectives 8.2 8.2 PrinciplePrinciple 8.3 8.3 InstrumentationInstrumentationSample Injection System Sample Injection System Column
Column
Packing Material or Stationary Phase Packing Material or Stationary Phase Solvent Supply System
Solvent Supply System Pumps
Pumps Detectors Detectors
8.4
8.4 Optimization of SeparationOptimization of Separation 8.5
8.5 AdvantagesAdvantages 8.6
8.6 Comparison with Gas ChromatograpComparison with Gas Chromatographyhy 8.7 8.7 ApplicationsApplications Polyaromatic Hydrocarbons Polyaromatic Hydrocarbons Isomeric Compounds Isomeric Compounds Sugars in Popular Drinks Sugars in Popular Drinks Drug Abuse
Drug Abuse
Separation of Nucleic Acids Separation of Nucleic Acids Analysis of Amino Acids Analysis of Amino Acids Partition Chromatography Partition Chromatography Ion Chromatography Ion Chromatography
Chiral Separation of Enantiomers Chiral Separation of Enantiomers Ion-Exclusion Chromatography Ion-Exclusion Chromatography Speciation Studies
Speciation Studies
8.8
8.8 Interfacing HPLC with Mass Interfacing HPLC with Mass SpectrometrySpectrometry
Thermospray Method Thermospray Method Particle Beam Interface Particle Beam Interface
Atmospheric Pressure Chemical Ionization Atmospheric Pressure Chemical Ionization Electrospray Interface
Electrospray Interface Moving Belt Interface Moving Belt Interface
8.9
8.9 SummarySummary 8.10
8.10 Terminal QuestionsTerminal Questions 8.11
8.11 AnswersAnswers
8.1
8.1
INTRODUCTION
INTRODUCTION
During early development period of column chromatography using a 50 - 100 c
During early development period of column chromatography using a 50 - 100 c m longm long and 1 - 5
and 1 - 5 cm diameter glass column packed with 100 - 200cm diameter glass column packed with 100 - 200 µµm particle size material, itm particle size material, it was realized that column efficiency was very low taking long time for analysis.
was realized that column efficiency was very low taking long time for analysis. Though, it could be increased by decreasing the column length and diameter and also Though, it could be increased by decreasing the column length and diameter and also the particle size of t
the particle size of t he column material. This could be made possible only after 1960he column material. This could be made possible only after 1960 when technology for produ
when technology for producing packing material with particle size of 3 cing packing material with particle size of 3 to 10to 10 µµm wasm was developed. Further, the new
developed. Further, the new technology required sophisticated instruments operatingtechnology required sophisticated instruments operating at high pressure contrary to classical system where eluent flows under gravity. The at high pressure contrary to classical system where eluent flows under gravity. The first instrument of liquid chromatograph was constructed by Csaba Horvath at first instrument of liquid chromatograph was constructed by Csaba Horvath at YaleYale University, USA in 1964 who describe it
University, USA in 1964 who describe it as high pressure liquid chromatographas high pressure liquid chromatograph (HPLC). However, he later called the technique as
(HPLC). However, he later called the technique as high performance liquid high performance liquid chromatography
chromatography . Thus, the new technique was named as “high pressure” or “high. Thus, the new technique was named as “high pressure” or “high performance” liquid chromatogra
performance” liquid chromatography (HPLC) to distinguish it phy (HPLC) to distinguish it from the old procedure.from the old procedure. Modern HPLC has emerged from the confluence of n
Modern HPLC has emerged from the confluence of n eed, the human desire eed, the human desire toto minimize work, technological capability and the
minimize work, technological capability and the theory to guide development alongtheory to guide development along rational lines. In some cases, HPLC may detect
It is now the
It is now the most versatile and widely used technique by chemists for the separation,most versatile and widely used technique by chemists for the separation, qualitative identification and quantitative determination of species in a variety of qualitative identification and quantitative determination of species in a variety of organic, inorganic, biologi
organic, inorganic, biological and other complex materials. It is cal and other complex materials. It is a type of elutiona type of elution chromatograp
chromatography where the sample, a mixture of solutes, is in hy where the sample, a mixture of solutes, is in a liquid solvent ora liquid solvent or mobile phase. The technique is
mobile phase. The technique is also known by other synonyms such asalso known by other synonyms such as high speedhigh speed chromatography
chromatography ,, high resolution chromatographyhigh resolution chromatography and and high efficiencyhigh efficiency chromatography
chromatography and is considered as the and is considered as the most sensitive method with continuousmost sensitive method with continuous major developments. HPLC is able
major developments. HPLC is able to separate macromolecules and ionic species,to separate macromolecules and ionic species, labile natural products, polymeric materials, and a wide variety of other high labile natural products, polymeric materials, and a wide variety of other high molecular weight polyfunctional groups. HPLC separations are based on specific molecular weight polyfunctional groups. HPLC separations are based on specific interactions between sample molecules with both the stationary and mobile phases. A interactions between sample molecules with both the stationary and mobile phases. A large variety of stationary phases available in HPLC allow a
large variety of stationary phases available in HPLC allow a great variety of selectivegreat variety of selective interactions causing better separations.
interactions causing better separations. Objectives
Objectives
After studying this Unit, you should be able to After studying this Unit, you should be able to
•• explain the explain the meaning of high performance liquid chromatography,meaning of high performance liquid chromatography,
•• differentiate between classical differentiate between classical liquid chromatography and HPLC,liquid chromatography and HPLC,
•• discuss the basic principle and discuss the basic principle and working of HPLC,working of HPLC,
•• describe various compodescribe various components of instrumentationents of instrumentation including n including stationary andstationary and mobile phases,
mobile phases,
•• describe characteristics of stationary phases in describe characteristics of stationary phases in various modes including bondedvarious modes including bonded phase,
phase,
•• know the solvent delivery system, characteristics of know the solvent delivery system, characteristics of mobile phase and elutionmobile phase and elution gradient,
gradient,
•• understand various detectors used in HPLC,understand various detectors used in HPLC,
•• learn about versatility and learn about versatility and advantages of HPLC,advantages of HPLC,
•• know about various interfaces while using mass know about various interfaces while using mass spectrometer as detector, andspectrometer as detector, and
•• learn about applications of HPLC for the analysis of a variety of solutes.learn about applications of HPLC for the analysis of a variety of solutes.
8.2
8.2
PRINCIPLE
PRINCIPLE
The basic principle of
The basic principle of separation by high performance liquid chromatography isseparation by high performance liquid chromatography is similar to classical
similar to classical liquid or column chromatography (LC) though it differs withliquid or column chromatography (LC) though it differs with regard to the size of the column
regard to the size of the column and the sample. It differs from LC in terms and the sample. It differs from LC in terms of speed,of speed, automation, elution time and individual manual assays of collected fractions. In case automation, elution time and individual manual assays of collected fractions. In case of HPLC, microgram amounts of the sample is allowed to pass through a column of HPLC, microgram amounts of the sample is allowed to pass through a column containing stationary solid inert phase coated with
containing stationary solid inert phase coated with nonvolatile liquid phase by meansnonvolatile liquid phase by means of pressurized flow of a
of pressurized flow of a liquid mobile phase where components migrate at differentliquid mobile phase where components migrate at different rates due to different relative affinities. Comparison of column size, characteristics of rates due to different relative affinities. Comparison of column size, characteristics of packing material and pressure requirements to force the
packing material and pressure requirements to force the mobility of mobile phase inmobility of mobile phase in classical column chromatography and HPLC are illustrated in
classical column chromatography and HPLC are illustrated in Fig. 8.1. According toFig. 8.1. According to another version, HPLC may be considered as
another version, HPLC may be considered as partition chromatography whpartition chromatography whereere stationary phase is a second liquid coated on an inert surface and it
stationary phase is a second liquid coated on an inert surface and it is immiscible withis immiscible with the liquid mobile phase.
the liquid mobile phase. According to the stationary liquid phase, According to the stationary liquid phase, the technique maythe technique may be subdivided into
be subdivided into two types; liquid-liquid and two types; liquid-liquid and liquid-bondliquid-bonded phase ed phase chromatograchromatography.phy. These differ from each other in the way stationary phase is held on
These differ from each other in the way stationary phase is held on to the supportto the support particles of the packing. In LLC, the polar liquid i
particles of the packing. In LLC, the polar liquid i s physically adsorbed on to an inerts physically adsorbed on to an inert surface where it competes with the mobile phase. However, in case of bonded phase surface where it competes with the mobile phase. However, in case of bonded phase chromatograp
(a)
(a) (b) (b) (c)(c)
Particle
Particle size: size: >150>150 µµm m 40-7040-70 µµm m 5-105-10 µµmm Column
Column diameter: diameter: 10-50 10-50 mm mm 1 1 – – 3 3 mm mm 2 2 – – 6 6 mmmm Column
Column length: length: 50-200 50-200 cm cm 50-100 50-100 cm cm 10-50 10-50 cmcm Pressure:
Pressure: < < 1 1 atm atm 30-50 30-50 atm atm 100-200 100-200 atmatm Fig. 8.1:
Fig. 8.1: Comparison of characteristics of various forms of liquid chromComparison of characteristics of various forms of liquid chromatography:atography: (a) Classical column chrom
(a) Classical column chromatography; atography; (b) HPLC with pel(b) HPLC with pellicular packing; (c)licular packing; (c) HPLC with microparticulate packing
HPLC with microparticulate packing
In order to
In order to achieve the desired separation by HPLC, several achieve the desired separation by HPLC, several operating conditionsoperating conditions including retention time, pressure and number of plates need to be optimized. A major including retention time, pressure and number of plates need to be optimized. A major interest is short analysis time, or
interest is short analysis time, or the plate count needed to accomplish a difficultthe plate count needed to accomplish a difficult separation. First of all,
separation. First of all, a proper HPLC system such aa proper HPLC system such as adsorption, bonded-phs adsorption, bonded-phase,ase, reverse phase, ion-exchange, exclusion, affinity or
reverse phase, ion-exchange, exclusion, affinity or any other form of any other form of chromatograpchromatographyhy must be selected. Then, all the
must be selected. Then, all the parameters in the equations as mentioned in Unit 4 thatparameters in the equations as mentioned in Unit 4 that depend on the properties of the
depend on the properties of the mobile and stationary phases are determined. Asmobile and stationary phases are determined. As already described in Unit 4, these are
already described in Unit 4, these are relative retentionrelative retention ((αα),), capacity factorcapacity factor ((k’k’) and) and the plate count
the plate count (( N N ). It is desired that ). It is desired that the compounds of interest should need at least tenthe compounds of interest should need at least ten times longer time to
times longer time to travel the column length than the unretained peak. Further, thetravel the column length than the unretained peak. Further, the viscosity of mobile phase and the diffusion coefficients of the solutes in the mobile viscosity of mobile phase and the diffusion coefficients of the solutes in the mobile phase are also
phase are also of concern besides other characteristics of of concern besides other characteristics of column packing.column packing. The plate height (
The plate height ( H H = = L L / / N N where, L where, L is the column is the column length andlength and N N the number of plates) is the number of plates) is reduced by the particle diameter (
reduced by the particle diameter (d d p p) and may be represented as) and may be represented as
h
h = = H H / / d d p p = = L L / / N N ..d d p p ... ... (8.1)(8.1)
It actually states the number of particle dia
It actually states the number of particle dia meter (meter (d d p p) that constitutes one plate height.) that constitutes one plate height.
Thus, reduced velocity may be
Thus, reduced velocity may be represented asrepresented as
vv = = u.d u.d p p / / D D M M … … (8.2)(8.2)
where,
where, D D M M is the diffusion coefficient of solute in the mobile phase. It is the diffusion coefficient of solute in the mobile phase. It may bemay be
considered as the
considered as the ratio of ratio of the time required to displace so the time required to displace solute molecules a distancelute molecules a distance equal to one particle diameter
equal to one particle diameter toto the time needed for the same displacement bythe time needed for the same displacement by molecular diffusion. It expresses the balance b
molecular diffusion. It expresses the balance b etween mass transport by diffusion oretween mass transport by diffusion or molecular motion across a single particle. Substituting the value of
molecular motion across a single particle. Substituting the value of uu (= (= L/t L/t mm), reduced), reduced
velocity may be expressed as velocity may be expressed as
vv == L L..d d p p /t /tmm.. D D M M … (8.3)… (8.3)
Thus, the complete equation for the dependence of the reduced plate height may be Thus, the complete equation for the dependence of the reduced plate height may be represented as modified van Deemter equation
represented as modified van Deemter equation h
h = = B/ B/ vv + + A.A.vv0..330..33 ++ C C ..vv … (8.4)… (8.4) where,
where, B B = 1.2 = 1.2 for solid core (pellicular) packing and 2.0 for solid core (pellicular) packing and 2.0 for completely porousfor completely porous column packing. Also,
column packing. Also, A A = 0 for well packed column and = 0 for well packed column and C C = 0.05 = 0.05 for porous particlesfor porous particles decreasing to 0.003 for pellicular particles.
decreasing to 0.003 for pellicular particles. No theory accurately describes theNo theory accurately describes the dispersion from flow in homogeneity in the
dispersion from flow in homogeneity in the mobile phase. A logarithmic plot mobile phase. A logarithmic plot ofof Eq. (8.4) is shown in Fig. 8.2. The reduced plate height has
Eq. (8.4) is shown in Fig. 8.2. The reduced plate height has a minimum value in thea minimum value in the range 2-3 for intermediate region of velocities where reduced velocity is 3 -5. range 2-3 for intermediate region of velocities where reduced velocity is 3 -5. It may be observed from Fig. 8.2 that
It may be observed from Fig. 8.2 that A A term dominates all a term dominates all a long whereaslong whereas B B term term arising from axial and longitudinal diffusion, dominates at
arising from axial and longitudinal diffusion, dominates at law reduced velocities. Thislaw reduced velocities. This region of
region of hh / / vv curve is usually avoided. At high velocities, however, curve is usually avoided. At high velocities, however, C C term responsible term responsible for increase in reduced plate height, dominates. As explained earlier,
for increase in reduced plate height, dominates. As explained earlier, C C term contains term contains the contributions from mass transfer kinetics and stagnant
the contributions from mass transfer kinetics and stagnant pockets of mobile phase.pockets of mobile phase. You can see that Eq. 8.4 r
You can see that Eq. 8.4 representing the reduced plate height is independent ofepresenting the reduced plate height is independent of particle diameter of
particle diameter of the column packing. The constantsthe column packing. The constants A A,, B B and and C C are dependent on are dependent on the packing of column. The number of plates in a reasonable time may be
the packing of column. The number of plates in a reasonable time may be optimizedoptimized while operating the
while operating the
Fig. 8.2:
Fig. 8.2: Logarithmic plot of reducLogarithmic plot of reduced plate height,ed plate height, h h against reduced velocity,against reduced velocity, νν with a with a
set of values of constants,
set of values of constants, A A = 1, = 1, B B = 2 and = 2 and C C = 0.1 = 0.1
column at the minimum in the
column at the minimum in the hh / / vv plot of Fig. 8.2. The co plot of Fig. 8.2. The column length and particle sizelumn length and particle size of the
of the t t mm and and N N are are chosen under the experimental conditions of eluent viscosity aschosen under the experimental conditions of eluent viscosity as
illustrated by the
illustrated by the following example.following example. Assuming desired plate counts,
Assuming desired plate counts, N N = 5000, reduced plate he = 5000, reduced plate height,ight, hh = 5 and a column = 5 and a column length,
length, L L = 250 mm, required plate diameter, = 250 mm, required plate diameter, d d p p may be calculated using Eq. (8.1). may be calculated using Eq. (8.1).
d
d p p == L L / / N N ..hh = 250/5000= 250/5000××5 5 = = 1/100 1/100 mm mm = = 1010 µµmm
Similarly, using viscosity parameter (
Similarly, using viscosity parameter (ηη) and specific column resistance () and specific column resistance (фф) for a fully) for a fully porous packing, pressure drop (
porous packing, pressure drop (∆∆PP) may be ) may be calculated using the expression.calculated using the expression.
2 2 22 3 3 M M p p M M LvD LvD NN hh P P d d t t φ φη η φφη η ∆ ∆ = = == … … (8.5)(8.5)
Combinations of column lengths and particle sizes
Combinations of column lengths and particle sizes including operating pressures forincluding operating pressures for different plate counts and retention times are available in literature.
different plate counts and retention times are available in literature. SAQ 1
SAQ 1
What are the various synonyms used for HPLC. Write each one of them. What are the various synonyms used for HPLC. Write each one of them.
………... ………... ………... ………...
8.3
8.3
INSTRUMENTATION
INSTRUMENTATION
General instrumentation for HPLC h
General instrumentation for HPLC h as following components.as following components. i)
i) One or more solvent reservoirs for the One or more solvent reservoirs for the mobile phase.mobile phase. ii)
ii) A pump to deliver the mobile phase with varying range of pressures up toA pump to deliver the mobile phase with varying range of pressures up to several hundred atmospheres to achieve reasonable flow rates.
several hundred atmospheres to achieve reasonable flow rates. iii)
iii) Sampling valves or loops where the sample may be injected into the flowingSampling valves or loops where the sample may be injected into the flowing mobile phase. Sample may be dissolved in mobile phase.
mobile phase. Sample may be dissolved in mobile phase. iv)
iv) A guard column or an on-line filter to prevent contamination of the mainA guard column or an on-line filter to prevent contamination of the main column.
column. v)
v) A pressure gauge, inserted in front A pressure gauge, inserted in front of the separation column, to of the separation column, to measure columnmeasure column inlet pressure.
inlet pressure. vi)
vi) Separation column containing packing to accomplish desired separation. TheseSeparation column containing packing to accomplish desired separation. These may be modified silica gel, ion-exchange resin, gel or some other unique may be modified silica gel, ion-exchange resin, gel or some other unique packing.
packing. vii)
vii) A detector capable enough of A detector capable enough of measuring the solute concentrations.measuring the solute concentrations. viii)
viii) Display and recording device for plotting time vs Display and recording device for plotting time vs peak intensity.peak intensity. Besides, other electronic accessories for
Besides, other electronic accessories for data manipulations are also required. Thesedata manipulations are also required. These are schematically shown in Fig. 8.3.
are schematically shown in Fig. 8.3.
Fig. 8.3:
The individual components are
The individual components are described below:described below: 8.3.1
8.3.1 Sample Injection SystemSample Injection System It is a l
It is a limiting factor in the precision of HPLC measurement because of reproducibilityimiting factor in the precision of HPLC measurement because of reproducibility with which samples may be
with which samples may be introduced onto the column packing. Insertion of theintroduced onto the column packing. Insertion of the sample into the
sample into the column must be through a narrow plug so column must be through a narrow plug so that peak broadening isthat peak broadening is minimized and the system
minimized and the system should have no dead volume by itself. should have no dead volume by itself. Generally, samplesGenerally, samples are dissolved in a mobile phase solvent to avoid solvent peak and 10 to
are dissolved in a mobile phase solvent to avoid solvent peak and 10 to 5050 µµL isL is introduced through micro sampling valves. These devices form an integral part introduced through micro sampling valves. These devices form an integral part ofof liquid chromatography equipment having interchangeable loops with a
liquid chromatography equipment having interchangeable loops with a choice ofchoice of sample size from 5 to 500
sample size from 5 to 500 µµL. The most widely uL. The most widely used method of sed method of introduction is bintroduction is basedased on sampling loop as shown in Fig. 8.4. It is
on sampling loop as shown in Fig. 8.4. It is filled by thoroughly flushing it using afilled by thoroughly flushing it using a
Fig. 8.4:
Fig. 8.4: Schematic of injector valve with external sample loop in a microvoSchematic of injector valve with external sample loop in a microvolume samplerlume sampler
sample solution by means of a microsyringe at pressures up to 7000 psi. A rotation of sample solution by means of a microsyringe at pressures up to 7000 psi. A rotation of the valve rotor places the sample filled
the valve rotor places the sample filled loop into the high pressure mobile phaseloop into the high pressure mobile phase stream whereby the sample is sent to the
stream whereby the sample is sent to the column. The system can be located within acolumn. The system can be located within a temperature controlled oven if handling at e
temperature controlled oven if handling at e levated temperatures is required. Manylevated temperatures is required. Many HPLC instruments incorpor
HPLC instruments incorporate an auto sampler with ate an auto sampler with an automatic injector that canan automatic injector that can inject variable volumes as
inject variable volumes as per requirement. In stopped flow injection method, pump isper requirement. In stopped flow injection method, pump is turned off till atmospheric pressure is attained, syringe is inserted and the sample turned off till atmospheric pressure is attained, syringe is inserted and the sample injected. The flow of sample can be brought to zero and rapidly resumed by diverting injected. The flow of sample can be brought to zero and rapidly resumed by diverting the mobile phase using a three way valve placed before the
the mobile phase using a three way valve placed before the injector. This method isinjector. This method is especially very useful for very high pressures. For best results, a two to fivefold excess especially very useful for very high pressures. For best results, a two to fivefold excess of sample should be passed through the
of sample should be passed through the loop to ensure that previous sample loop to ensure that previous sample has beenhas been purged thoroughly.
purged thoroughly. 8.3.2
8.3.2 ColumnColumn It is the heart
It is the heart of the HPLC instrumentof the HPLC instrument where actual separation occurs. Separationwhere actual separation occurs. Separation column in HPLC is usually made of heavy wall, glass lined metal
column in HPLC is usually made of heavy wall, glass lined metal or 316 gradeor 316 grade stainless steel tubing, that can withstand high pressure and which is inert t stainless steel tubing, that can withstand high pressure and which is inert t o theo the chemical corrosion due to mobile phase. The interior of the tubing must be smooth chemical corrosion due to mobile phase. The interior of the tubing must be smooth with a uniform bore diameter. Straight columns that can be operated in vertical with a uniform bore diameter. Straight columns that can be operated in vertical position are preferred. Some typical tubing materials used in HPLC column are listed position are preferred. Some typical tubing materials used in HPLC column are listed in Table 8.1.
Table 8.1:
Table 8.1: Column Tubing Column Tubing Materials and its UseMaterials and its Usess
Material Use
Material Use
316
316 Stainless Stainless steel steel (SS) (SS) General General utility utility material, material, good good for for highhigh pressure system
pressure system Poly (ether-ether) ketone
Poly (ether-ether) ketone (PEEK)
(PEEK)
Inert to most organic solvents except Inert to most organic solvents except methylene chloride, THF, DMSO and conc. methylene chloride, THF, DMSO and conc. Sulphuric and nitric acids. Holds
Sulphuric and nitric acids. Holds pressurespressures up to 5000 psi (34MPa).
up to 5000 psi (34MPa).
Good for metal-free biological systems. Good for metal-free biological systems. Tefzel
Tefzel Inert. Inert. Common Common for for metal-free metal-free applications.applications. Titanium
Titanium Withstands Withstands pressures pressures up up to to 5000 5000 psi,psi, corrosion resistant; expensive corrosion resistant; expensive Fused silica Glass
Fused silica Glass Glass
Glass
Used for capillary LC. Used for capillary LC. Limited pressure range. Limited pressure range. Glass-lined
Glass-lined SS SS Inert, Inert, withstands withstands pressures pressures but but difficult difficult toto know when the glass is
know when the glass is broken.broken.
Column fittings and connectors must be
Column fittings and connectors must be so designed that void so designed that void volume is zero avoidingvolume is zero avoiding unswept corners. Column length ranges
unswept corners. Column length ranges10 to 30 cm with 10 to 30 cm with inner diameter of 2 to 5 mminner diameter of 2 to 5 mm providing 40,000 to 60,000 plates per inch. However, shorter columns of
providing 40,000 to 60,000 plates per inch. However, shorter columns of 3 to 8 3 to 8 cm arecm are also used for fast separations but in such cases, sample size
also used for fast separations but in such cases, sample size will become limited. Thewill become limited. The length of the column may not only affect the resolution of a
length of the column may not only affect the resolution of a given separation –thegiven separation –the longer the column the larger number of plates but also the
longer the column the larger number of plates but also the speed of separation.speed of separation. Standard lengths vary with the
Standard lengths vary with the manufacturer but most common values are 30, 25, manufacturer but most common values are 30, 25, 15,15, 12.5, 10 and 7.5 cm. It may be noted
12.5, 10 and 7.5 cm. It may be noted that shorter columns are described as high speedthat shorter columns are described as high speed columns. The columns packed with the finer particles are more expensive than the columns. The columns packed with the finer particles are more expensive than the standard 5 µm packing.
standard 5 µm packing. Guard column:
Guard column:In order to increase the life of In order to increase the life of analytical column, a short guardanalytical column, a short guard column, also called
column, also called precolumn precolumn, is placed before the main , is placed before the main column as shown in Fig. 8.3.column as shown in Fig. 8.3. It removes contamination from the solvent. Guard
It removes contamination from the solvent. Guard column serves to saturate thecolumn serves to saturate the mobile phase with the stationary phase so that losses of
mobile phase with the stationary phase so that losses of stationary phase in the columnstationary phase in the column are minimized. However, it is essential t
are minimized. However, it is essential t hat the composition of the guard columnhat the composition of the guard column should be similar to that of the
should be similar to that of the analytical column but its particle size may be analytical column but its particle size may be larger tolarger to minimize the pressure drop.
minimize the pressure drop. 8.3.3
8.3.3 Packing Material or Stationary PhasePacking Material or Stationary Phase
The packing used in modern HPLC consists of small, rigid particles having a narrow The packing used in modern HPLC consists of small, rigid particles having a narrow particle size distribution. The most common packing material used for LC is prepared particle size distribution. The most common packing material used for LC is prepared from silica (acidic) and alumina (basic) particles which are synthesized by
from silica (acidic) and alumina (basic) particles which are synthesized by agglomerating submicron size particles under conditions that lead
agglomerating submicron size particles under conditions that lead to larger particleto larger particle diameter. These are often coated with thin organic films which are physically or diameter. These are often coated with thin organic films which are physically or chemically bonded to the surface. For nearly all HPLC applications, chemically chemically bonded to the surface. For nearly all HPLC applications, chemically modified or unmodified micro particulate silicas of 3, 5 or 10
modified or unmodified micro particulate silicas of 3, 5 or 10 µµm diameter arem diameter are preferred. This form of LLC,
preferred. This form of LLC, in which both monomeric and polymeric phases havein which both monomeric and polymeric phases have been bonded to a wide range of support materials, is called
been bonded to a wide range of support materials, is called bonded phasebonded phase chromatography
chromatography (BPC). Characteristics of typical packing materials used in HPLC are (BPC). Characteristics of typical packing materials used in HPLC are listed in Table 8.2.
listed in Table 8.2. The particles used in HPLC, which are totally porousThe particles used in HPLC, which are totally porous (macroporou
(macroporous) or s) or superficially porous (pellicular) support, may be superficially porous (pellicular) support, may be spherical orspherical or irregular in shape but it is
Table 8.2:
Table 8.2: Characteristics of Some Characteristics of Some Commercial HPLC Commercial HPLC Column PackingColumn Packing Materials
Materials
Type
Type Pellicular Pellicular MicroprousMicroprous
Silica
Silica Corasil (37-50Corasil (37-50µµm) m) Lichrosorb (5,10 Lichrosorb (5,10 & & 2020 µµm)m) Vydac (30-40
Vydac (30-40µµm) m) Micropak (5&10Micropak (5&10 µµm)m) Porasil (15 & 20 Porasil (15 & 20 µµm)m) Spherisorb (5
Spherisorb (5 µµm)m) Alumina
Alumina Perisorb PA Perisorb PA Micropak Al Micropak Al (5 (5 & & 1010 µµm)m) Spherisorb Al
Spherisorb Al
ensure high column efficiency and permeability. These adsorbent packings retain ensure high column efficiency and permeability. These adsorbent packings retain solute molecules almost exclusively on the internal surface of the pores, thus, solute molecules almost exclusively on the internal surface of the pores, thus, separating these from others. Various types of bonded phases used
separating these from others. Various types of bonded phases used in HPLC arein HPLC are schematically shown in Fig. 8.5.
schematically shown in Fig. 8.5.
Fig. 8.5: Various shapes of stationary phase packings used in HPLC: Fig. 8.5: Various shapes of stationary phase packings used in HPLC:
(a) Bonded (spherical) phase; (b) Irregular large porous phase; (a) Bonded (spherical) phase; (b) Irregular large porous phase; (c) Pellicular particle beads and (d) Porous microparticle (c) Pellicular particle beads and (d) Porous microparticle
The characteristics of various types of
The characteristics of various types of bonded phases are described below:bonded phases are described below: A.
A. Spherical bonded phase:Spherical bonded phase: These spherical packings consist of a solid, sphericalThese spherical packings consist of a solid, spherical nonporou
nonporous core (usually a s core (usually a glass bead) with a glass bead) with a layer of attached functional groupslayer of attached functional groups forming an outer shell containing unmodified or modified silica
forming an outer shell containing unmodified or modified silica gel, resin,gel, resin, polyamide,
polyamide, etc.etc. Various functional groups are used depending on the Various functional groups are used depending on the nature ofnature of solutes to be separated.
solutes to be separated. B.
B. Porous layer beads:Porous layer beads:A porous or pellicular layer A porous or pellicular layer bead type packing materialbead type packing material consists of a solid, spherical with an a
consists of a solid, spherical with an a verage particle diameter 30-40verage particle diameter 30-40 µµm coatedm coated with a thin porous outer shell, typically of 1-3
with a thin porous outer shell, typically of 1-3 µµm thick. It may be a silica gelm thick. It may be a silica gel layer, a network of small spherical particles bonded to the solid core.
layer, a network of small spherical particles bonded to the solid core. It may alsoIt may also be monomeric or polymeric organic phase. Surface areas
be monomeric or polymeric organic phase. Surface areas of the porous layerof the porous layer beads range from 5 to 15 m
beads range from 5 to 15 m22 /g . These materials are easy /g . These materials are easy to be packed becauseto be packed because of its dense core but suffer from limited sample capacity due
of its dense core but suffer from limited sample capacity due to small surfaceto small surface areas. Porous layer packings exhibit good efficiency because of
areas. Porous layer packings exhibit good efficiency because of improved massimproved mass transfer within the stationary phase.
transfer within the stationary phase. Longer columns are possible because theLonger columns are possible because the pressure drop is lower due to larger particle size of porous layer supports. pressure drop is lower due to larger particle size of porous layer supports. Thicker coatings give rise to slower mass transfer but have increased sample Thicker coatings give rise to slower mass transfer but have increased sample capacity.
capacity. C.
C. Porous particles:Porous particles: Totally porous particles have a large surface area in the Totally porous particles have a large surface area in the rangerange 100 to 860 m
100 to 860 m22 /g with averag /g with average being 400 me being 400 m22 /g. The mean por /g. The mean pore diameter ise diameter is inversely related to the specific surface area where small molecules enter inversely related to the specific surface area where small molecules enter thethe pores. The particles can be packed into the HPLC
pores. The particles can be packed into the HPLC column of efficiencies up tocolumn of efficiencies up to 800 theoretical plates per centimeter if 5
larger particles give proportionately small number of theoretical plates
larger particles give proportionately small number of theoretical plates whencewhence the efficiency of separation goes down.
the efficiency of separation goes down. D.
D. Macroporous particles:Macroporous particles: These are recently introduced graphitized carbon andThese are recently introduced graphitized carbon and styrene-diviny
styrene-divinylbenzene polymers having large channels blbenzene polymers having large channels b esides micropores. Theesides micropores. The rigid, porous polymeric macroporo
rigid, porous polymeric macroporous beads do not us beads do not swell or shrink with swell or shrink with changeschanges in the ionic strength of the mobile phase
in the ionic strength of the mobile phase (can be used over an extended pH range(can be used over an extended pH range of 1 and 13) or deform at high velocities and are
of 1 and 13) or deform at high velocities and are most suited for separations inmost suited for separations in nonaqeous media. These materials have increased the
nonaqeous media. These materials have increased the choice of stationarychoice of stationary phases and the scope of HPLC, particularly for highly polar and basic phases and the scope of HPLC, particularly for highly polar and basic substances.
substances.
An illustration of various types of bonded phases used in HPLC is shown in Fig. 8.6 An illustration of various types of bonded phases used in HPLC is shown in Fig. 8.6 where different topographies are obtained depending on the nature of
where different topographies are obtained depending on the nature of the ligand . the ligand . ItIt may be noted that different packing materials are used in different type of techniques may be noted that different packing materials are used in different type of techniques of adsorption, partition,
of adsorption, partition, ion-exchaion-exchange, size nge, size exclusion chromatographyexclusion chromatography..
(a)
(a) (b) (b) (c)(c)
Fig. 8.6:
Fig. 8.6: Various shapVarious shapes of bonded HPLC es of bonded HPLC column packing materialscolumn packing materials (a) Type
(a) Types of bos of bonded phases, nded phases, (b) Top(b) Topography of ography of ligands ligands and (c) and (c) Size of ligSize of ligandsands
SAQ 2 SAQ 2
Explain why small particle size is required in HPLC?
Explain why small particle size is required in HPLC? How is it important in attainingHow is it important in attaining higher efficiency? higher efficiency? ………... ………... ………... ………... ………... ………... SAQ 3 SAQ 3
Choose the correct answer from the
Choose the correct answer from the choices given.choices given. i)
i) Which one of the following is the most appropriate particle size (in µWhich one of the following is the most appropriate particle size (in µ m) form) for packing material in HPLC?
packing material in HPLC? a)
a) 1-5 1-5 b) b) 3-5 3-5 c) c) 10-20 10-20 d) d) 20-5020-50 ii)
ii) Which one of the following column length (in cm) should be used Which one of the following column length (in cm) should be used for fasterfor faster HPLC separation?
HPLC separation? a)
iii)
iii) Which of the following materials meet the requirements to fabricate HPLCWhich of the following materials meet the requirements to fabricate HPLC column?
column? a) Glass
a) Glass lined lined metal metal b) Quartz b) Quartz c) Stainless c) Stainless steel steel d) Steeld) Steel iv)
iv) What is the average surface area ( What is the average surface area ( in min m22 /g) of porou /g) of porous particles in HPLC colus particles in HPLC column?mn? a)
a) 100 100 b) b) 300 300 c) 800 c) 800 d) d) 400400 v)
v) Which one of the Which one of the following ranges of flow rates (in following ranges of flow rates (in mL/min) should be adequatemL/min) should be adequate for analytical HPLC?
for analytical HPLC? a)
a) 0.02 0.02 – – 1.0 1.0 b) b) 0.05 0.05 - - 2.0 2.0 c) c) 1.0-2.0 1.0-2.0 d) d) 0.5 0.5 – – 2.02.0 Let us now study about the s
Let us now study about the stationary phasestationary phases used in used in various chromatogvarious chromatographic modes.raphic modes. i)
i) Adsorption ChromatographyAdsorption Chromatography In majority of the
In majority of the cases of adsorption chromatographycases of adsorption chromatography, silica , silica column packingscolumn packings are used where main mechanism is the interaction of its
are used where main mechanism is the interaction of its OH groups with theOH groups with the polar or unsaturated functional groups of a solute/solvent molecule by
polar or unsaturated functional groups of a solute/solvent molecule by hydrogenhydrogen bonding or dipole interaction. The slightly acidic
bonding or dipole interaction. The slightly acidic silanol (Si-OH) groups insilanol (Si-OH) groups in silica gel are at
silica gel are at the surface and extend out from the surface in the the surface and extend out from the surface in the internalinternal channels of the pore structure.
channels of the pore structure. The number and topographical arrangement ofThe number and topographical arrangement of the several types of OH groups, as shown in Fig. 8.7, determine the activity of the several types of OH groups, as shown in Fig. 8.7, determine the activity of the adsorbent and thereby the retention of the solutes. These OH groups can be the adsorbent and thereby the retention of the solutes. These OH groups can be divided into three types:
divided into three types:
•• silanol (free OH),silanol (free OH),
•• siloxane bond (Si-O-Si) andsiloxane bond (Si-O-Si) and
•• hydrogen bond (Si-OH…O).hydrogen bond (Si-OH…O).
Fig. 8.7:
Fig. 8.7: Structure of silica gel depicting the various types of hydroxyl groupStructure of silica gel depicting the various types of hydroxyl groups thats that interact with the functional groups of solute/solvent molecules
interact with the functional groups of solute/solvent molecules
Each of these groups has different activity that increases in the
Each of these groups has different activity that increases in the following order:following order: Bound
Bound < < free free < < H-bond.H-bond.
According to current models of adsorption process, it
According to current models of adsorption process, it is assumed that adsorptionis assumed that adsorption sites are completely covered by either of solute or solvent molecules that are sites are completely covered by either of solute or solvent molecules that are adsorbed depending on their relative strength in
competition between the solute and the mobile phase molecules for an active competition between the solute and the mobile phase molecules for an active site provides the driving force and
site provides the driving force and selectivity in separations. Interaction betweenselectivity in separations. Interaction between a solute molecule and
a solute molecule and the adsorbent surface is best the adsorbent surface is best when functional groupswhen functional groups overlap adsorption sites. Adsorption chromatography is less
overlap adsorption sites. Adsorption chromatography is less influenced byinfluenced by difference in molecular weight but certainly
difference in molecular weight but certainly more by functional groupsmore by functional groups. For. For compounds of low to
compounds of low to moderate polarity, adsorption chromatograpmoderate polarity, adsorption chromatography oftenhy often makes possible the separation of
makes possible the separation of complex mixtures into classes of compoundscomplex mixtures into classes of compounds with similar chemical
with similar chemical functionality. Typical examples of group separations arefunctionality. Typical examples of group separations are polynuclear aromatics from a petroleum sample and the
polynuclear aromatics from a petroleum sample and the triglycerides from atriglycerides from a liquid extract.
liquid extract. ii)
ii) Partition ChromatographyPartition Chromatography It can be subdivided into
It can be subdivided into liquid-liquid liquid-liquid chromatogrchromatographyaphy (LLC) and (LLC) and bonded phasebonded phase chromatography
chromatography (BPC), the difference being in (BPC), the difference being in the method by which stationarythe method by which stationary phase is held on the support particles of the packing. In case
phase is held on the support particles of the packing. In case of LLC, a liquidof LLC, a liquid stationary phase is retained on
stationary phase is retained on the surface of the pthe surface of the p acking by physical adsorption.acking by physical adsorption. With bonded phase, the stationary phase is bonded chemically to the surface of With bonded phase, the stationary phase is bonded chemically to the surface of inert support. Of late bonded phase has
inert support. Of late bonded phase has become predominant over liquid phasebecome predominant over liquid phase because of certain disadvantages. The packings for bonded phase are
because of certain disadvantages. The packings for bonded phase are preparedprepared from rigid silica or silica based compositions. These are formed as uniform, from rigid silica or silica based compositions. These are formed as uniform, porous, mechanically sturdy particles commonly having diameters 3, 5 or 10 porous, mechanically sturdy particles commonly having diameters 3, 5 or 10 µ
µm. The surface of fully hydrolysed silica is made up of chemically silanolm. The surface of fully hydrolysed silica is made up of chemically silanol groups. The most useful bonded phase coatings are
groups. The most useful bonded phase coatings are siloxanes formed by thesiloxanes formed by the reaction of hydrolysed surface with an organochlorosilane as shown below: reaction of hydrolysed surface with an organochlorosilane as shown below:
S Sii OOHH CCll SSii RR CH CH33 CH CH33 Si Si OO SiSi RR CH CH33 CH CH33 + + ++ HClHCl Si Si OHOH CCll SSii ClCl Si Si OHOH Si Si OO SiSi OO SSii CCll CH CH33 CH CH33 CH CH33 CH CH33 Si Si OO SiSi OO SSii CCll CH CH33 CH CH33 CH CH33 CH CH33 CH CH33 + + HH22OO
Surface coverage by silanization is limited to 4
Surface coverage by silanization is limited to 4 µµmol/mmol/m22 or less because of steric or less because of steric effects. The unreacted SiOH groups impart an undesirable polarity to
effects. The unreacted SiOH groups impart an undesirable polarity to thethe surface, which may lead to
surface, which may lead to chromatograchromatographic tailing of phic tailing of the peaks. In order tothe peaks. In order to avoid this effect, siloxane packings are
avoid this effect, siloxane packings are often capped by further reaction withoften capped by further reaction with chloromethyl
chloromethylsilane that can react with many of the unreacted silane that can react with many of the unreacted silanol groups.silanol groups. Two types of partition chromatography have been recognized based on relative Two types of partition chromatography have been recognized based on relative polarities of stationary phase and mobile phase. In normal phase LC or HPLC, polarities of stationary phase and mobile phase. In normal phase LC or HPLC, stationary phase consists of highly polar water
stationary phase consists of highly polar water or triethyleneglycol supported onor triethyleneglycol supported on silica or alumina particles and a
silica or alumina particles and a nonpolar mobile phase solvenonpolar mobile phase solvent such as hexane isnt such as hexane is used. In contrast, in
used. In contrast, in the reversed phase chromatographythe reversed phase chromatography, the , the stationary phase isstationary phase is nonpolar, often a hydrocarbon and the mobile phase is
nonpolar, often a hydrocarbon and the mobile phase is polar such as polar such as water,water, methanol or acetonitrile where most
methanol or acetonitrile where most the polar component appears first. Perhapsthe polar component appears first. Perhaps three quarters of all the HPLC is
three quarters of all the HPLC is currently being carried out in columns withcurrently being carried out in columns with reversed phase.
Most commonly, the R group of the siloxane in these coatings is a
Most commonly, the R group of the siloxane in these coatings is a nn-octyl (C-8-octyl (C-8 chain) or
chain) or nn-octadecyl (C-18 chain). With such preparations, the -octadecyl (C-18 chain). With such preparations, the long chainlong chain hydrocarbo
hydrocarbon groups are aligned parallel n groups are aligned parallel to one another and perpendicular to to one another and perpendicular to thethe particle surface, giving a brush or bristle-like structure as illustrated in Fig. 8.6. particle surface, giving a brush or bristle-like structure as illustrated in Fig. 8.6. The relationship the between polarity of the sample with that of
The relationship the between polarity of the sample with that of the columnthe column packing material and mobile phase is illustrated in
packing material and mobile phase is illustrated in Fig. 8.8. Retention increasesFig. 8.8. Retention increases with the hydrophobic character of the solute samples. Generally, the lower the with the hydrophobic character of the solute samples. Generally, the lower the polarity of the mobile phase, the higher is i
polarity of the mobile phase, the higher is i ts eluent strength. The effect of chaints eluent strength. The effect of chain length of the alkyl group upon column performa
length of the alkyl group upon column performance is illustrated in nce is illustrated in Fig. 8.8Fig. 8.8 where it is observed that longer chains produce packings that are more retentive. where it is observed that longer chains produce packings that are more retentive. For example, maximum sample size for a C
For example, maximum sample size for a C1818packing is roughly double that forpacking is roughlydouble that for
a C
a C44 preparation under similar preparation under similar experimental conditions.experimental conditions.
Fig. 8.8:
Fig. 8.8: Relationship between the poRelationship between the polarity of the sample with that of the packinglarity of the sample with that of the packing material and the mobile phase in reverse phase HPLC
material and the mobile phase in reverse phase HPLC
In commercial normal-phase bonded packings, the R in the
In commercial normal-phase bonded packings, the R in the siloxane structure issiloxane structure is a polar functional group such as
a polar functional group such as cyano (−Ccyano (−C22HH44CN), diolCN), diol
(–C
(–C33HH66OCHOCH22CHOHCHCHOHCH22OH), amino (−COH), amino (−C33HH66NHNH22), and d), and dimethylaminoimethylamino
(C
(C33HH66N(CHN(CH33))22). The polarities of these packing materials vary over a). The polarities of these packing materials vary over a
considerable range with the cyano type being the last polar and the amino types considerable range with the cyano type being the last polar and the amino types the most. Diol packings are intermediate in polarity. With normal phase
the most. Diol packings are intermediate in polarity. With normal phase packings, elution is carried with
packings, elution is carried with relatively non-polar solvents such as ethylrelatively non-polar solvents such as ethyl ether, chloroform and
ether, chloroform and nn-hexane.-hexane. iii)
iii) Ion-exchange ChromatographyIon-exchange Chromatography In this case,
In this case, column packings have charge bearing functional groups attached tocolumn packings have charge bearing functional groups attached to a polymer matrix. The
a polymer matrix. The functional groups are permanently bonded ionic groupsfunctional groups are permanently bonded ionic groups associated with counterions of the opposite charge. Some
associated with counterions of the opposite charge. Some ion-exchangion-exchangee packings bear negatively charged groups and are used for
packings bear negatively charged groups and are used for exchanging cationicexchanging cationic species whereas others are designed for
species whereas others are designed for exchanging anionic species. Similarly,exchanging anionic species. Similarly, some functional groups such as –COOH or
some functional groups such as –COOH or -PO-PO332–2–have weak acidic or basichave weak acidic or basic
properties whereas some others have considerable affinity for heavy metal properties whereas some others have considerable affinity for heavy metal cations. Several structural types of packings, as shown in Fig. 8.9, have been cations. Several structural types of packings, as shown in Fig. 8.9, have been used in ion-exchange HPLC.
used in ion-exchange HPLC. Of these, the
Of these, the pellicular type consists pellicular type consists of a resin coating, aboof a resin coating, about 1-2ut 1-2 µµm thick, on am thick, on a glass bead of 30-40
glass bead of 30-40 µµm diameter. Superficially porous resins are obtained bym diameter. Superficially porous resins are obtained by coating glass beads with a thin layer of
coating glass beads with a thin layer of silica microspheres on which ionsilica microspheres on which ion exchanger is bonded. This increases the interface between the resin and mobile exchanger is bonded. This increases the interface between the resin and mobile phase. Either type of these packings have low exchange capacity, 0.01 – 0.1 phase. Either type of these packings have low exchange capacity, 0.01 – 0.1 meq/g. The exchanger may also be bonded to silica microparticles by means of meq/g. The exchanger may also be bonded to silica microparticles by means of silylation reactions or polymerized into pores of
(a)
(a) (b) (b) (c) (c) (d)(d)
Fig. 8.9:
Fig. 8.9: Various structural types Various structural types of ion-exchange paof ion-exchange packings: (a) pellicular withckings: (a) pellicular with
ion-exchange film; (b) exchanger beads coated superficially with porous resin; ion-exchange film; (b) exchanger beads coated superficially with porous resin; (c) macroreticular resin bead and (d) anion exchanger surface sulfonated and (c) macroreticular resin bead and (d) anion exchanger surface sulfonated and bonded electrostatically
bonded electrostatically
During preparation of ion exchanger by silylation, a
During preparation of ion exchanger by silylation, a vinyl group is chosen for Rvinyl group is chosen for R33
in -SiOSiR
in -SiOSiR11RR22RR33leading to a vinylated silica which is leading to a vinylated silica which is then polymerized withthen polymerized with
styrene. styrene. CH CH CHCH22 CHCH CHCH22 CHCH CHCH22 CHCH CHCH22 C C66HH55 CC66HH55 = = ++ ==
Afterwards, the bonded phase is treated with
Afterwards, the bonded phase is treated with chloromethyl ether andchloromethyl ether and subsequently tr
subsequently trimethylamine imethylamine or hydroxyethyor hydroxyethyldimethylamine to prepare theldimethylamine to prepare the quaternary amine exchanger as is shown below:
quaternary amine exchanger as is shown below:
CH CH22 CHCH22 CCHH CCHH22 ClCHClCH22OCHOCH33 CHCH22 CHCH22 CHCH CHCH22 CHCH33OHOH C C66HH55CHCH22ClCl C C66HH55 CH CH22 CHCH22 CHCH CHCH22 C C66HH55CHCH22NHNH22-R-R CH CH22 CHCH22 CHCH CHCH22 C C66HH55CHCH22N(CHN(CH33))33 N(CH N(CH33))22CHCH22CHCH22OHOH RNH RNH22 + + ++ + +
Weak anion exchanger
Weak anion exchanger Strong anion exchangerStrong anion exchanger +
+
Hydrop
Hydrophilic polymers allow the hilic polymers allow the separation of proteins, nucleic acids and otherseparation of proteins, nucleic acids and other large ionic molecules. The microporosity of these
large ionic molecules. The microporosity of these ion exchangers minimizesion exchangers minimizes possible exclusion effects.
possible exclusion effects. iv)
iv) Size Exclusion ChromatographySize Exclusion Chromatography In this case,
In this case, column packings are either semi-rigid, cross-linked macromolecularcolumn packings are either semi-rigid, cross-linked macromolecular polymers or rigid, controlled pore size glasses or silicas. The semi-rigid
polymers or rigid, controlled pore size glasses or silicas. The semi-rigid materials swell and care must be
materials swell and care must be taken to their use limited to a taken to their use limited to a maximummaximum pressure of 300 psi due
pressure of 300 psi due to bed compressibility. The styrene-divinyl benzeneto bed compressibility. The styrene-divinyl benzene polymers allow fractionation within a molecular weight range of
polymers allow fractionation within a molecular weight range of 100 to 5000100 to 5000 million. Partially sulphonated polystyrene beads are compatible with aqueous million. Partially sulphonated polystyrene beads are compatible with aqueous systems and non-sulphonated ones with non-aqueous systems with
systems and non-sulphonated ones with non-aqueous systems with beadbead diameters ~5
diameters ~5 µµm.m.
Another class of hydrophilic porous packing is prepared by suspension Another class of hydrophilic porous packing is prepared by suspension polymerization of 2-hydroxy
polymerization of 2-hydroxyethyl methacrylate with ethyl methacrylate with ethylene dimethacrylate.ethylene dimethacrylate. These packings can withstand pressures up to 3000 psi and are usable with These packings can withstand pressures up to 3000 psi and are usable with aqueous systems and with a
aqueous systems and with a variety of polar organic solvents. Porous glasses andvariety of polar organic solvents. Porous glasses and silicas cover a wide range of pore size
silicas cover a wide range of pore size diameters. For example, a series ofdiameters. For example, a series of particle size diameters and operating ranges of molecular weights are listed in particle size diameters and operating ranges of molecular weights are listed in Table 8.3.
Table 8.3: Correlation of Pore Size Diameter and Operating Range of Mol. Wt Table 8.3: Correlation of Pore Size Diameter and Operating Range of Mol. Wt
Pore-size diameter Pore-size diameter ((
µµ
m)m) Operating range Operating range (Daltons) (Daltons) 4 1000-8000 4 1000-8000 10 1000-30000 10 1000-30000 25 2500-125000 25 2500-125000 55 11000-350000 55 11000-350000 150 100000-1000000 150 100000-1000000 250 200000-1500000 250 200000-1500000These packings are chemically resistant at pH <10 and can be u
These packings are chemically resistant at pH <10 and can be u sed with aqueoussed with aqueous and polar
and polar organic solvents. With nonorganic solvents. With nonpolar solvents, it is desirable to deactivatepolar solvents, it is desirable to deactivate the surface by silylation.
the surface by silylation. Porous inorganic packingPorous inorganic packings have distinct s have distinct advantagesadvantages over organic exclusion packings. The surface of a
over organic exclusion packings. The surface of a typical hydrophilic packingtypical hydrophilic packing has the following structure;
has the following structure;
S
Sii CCHH22 CHCH22 CHCH22 OO CHCH22 CHCH22 CC CHCH22OHOH H
H
Columns can be used
Columns can be used routinely and indefinitely after calibration, without anyroutinely and indefinitely after calibration, without any possibility of sample contamination or biodegradation. Properties of some possibility of sample contamination or biodegradation. Properties of some commercial size exclusion packings are listed in Table 8.4.
commercial size exclusion packings are listed in Table 8.4. Table 8.4:
Table 8.4: Properties of TypicProperties of Typical Commercial Packinal Commercial Packings for Size-Exclusiongs for Size-Exclusion Chromatography Chromatography Type Particle Type Particle Size, Size,
µµ
mm Average Pore Average Pore Size, Size,Ǻ
Ǻ
Molecular eight Molecular eight Exclusion Limit Exclusion Limit Polystyrene-divinylbenzene divinylbenzene 10 10 10 1022 700700 10 1033 (0.1 (0.1 – – 20) 20) × × 101044 10 1044 (1 (1 – – 20) 20) × × 101044 10 1055 (1 (1 – – 20) 20) × × 101055 10 1066 (5 (5 – – >10) >10) × × 101066 Silica Silica 10 10 125 125 (0.2 (0.2 – – 5) 5) × × 101044 300 300 (0.03 (0.03 – – 1) 1) × × 101055 500 500 (0.05 (0.05 – – 5) 5) × × 101055 1000 1000 (5 (5 – – 20) 20) × × 101055 v)v) Ion ChromatographyIon Chromatography
It differs from ion-exchange chromatography in the nature of exchange resins. It differs from ion-exchange chromatography in the nature of exchange resins. The technique involves an ion-exchange column and a means
The technique involves an ion-exchange column and a means of suppressingof suppressing (removing) ionic species other than the sample ions in the eluting mobile phase (removing) ionic species other than the sample ions in the eluting mobile phase to facilitate detection of the
to facilitate detection of the sample by a conductivity monitor as schematicallysample by a conductivity monitor as schematically illustrated in Fig. 8.10.
Fig. 8.10:
Fig. 8.10: Schematic diagram of ion chSchematic diagram of ion chromatograph with separomatograph with separation columnration column
The column packing consists of a neutral polymer core of ~ 10
The column packing consists of a neutral polymer core of ~ 10 µµm diameterm diameter depending on whether the packing will be used for the separation of cations or depending on whether the packing will be used for the separation of cations or anions. Contrary to
anions. Contrary to the conventional ion-exchange chromatograthe conventional ion-exchange chromatography where corephy where core is sulphonated or aminated leading to the formation of sulfonic acid or
is sulphonated or aminated leading to the formation of sulfonic acid or
quaternary amine groups, in ion chromatography, a monolayer of aminated or quaternary amine groups, in ion chromatography, a monolayer of aminated or sulphonated polymeric anion exchange beads is
sulphonated polymeric anion exchange beads is used.used.
Similarly, for a cation exchanger, there would be an intermediate layer of Similarly, for a cation exchanger, there would be an intermediate layer of aminated groups covered by a thin
aminated groups covered by a thin layer of sulphonated resin beads. Due to layer of sulphonated resin beads. Due to thethe proximity of all the active sites t
proximity of all the active sites to the eluent-resin interface, this type ofo the eluent-resin interface, this type of exchanger has favor
exchanger has favorable mass transfer characteristics. able mass transfer characteristics. It has low exchangeIt has low exchange capacity, about 0.020 meq/g of copolymer. In most
capacity, about 0.020 meq/g of copolymer. In most applications, silica basedapplications, silica based materials are inappropriate due to
materials are inappropriate due to their degradation in the presence of their degradation in the presence of aqueousaqueous eluents and their poor selectivity for some ionic species. The eluent passes eluents and their poor selectivity for some ionic species. The eluent passes through a suppressor column where the eluting or background electrolyte is through a suppressor column where the eluting or background electrolyte is effectively removed by converting it into water
effectively removed by converting it into water or, water and carbon dioxideor, water and carbon dioxide ii..ee.,., sodium ions are replaced by hydronium ions or
sodium ions are replaced by hydronium ions or methylsulfomethylsulfonate ions withnate ions with hydroxyl ions.
hydroxyl ions. A miniaturized
A miniaturized ‘self-regenerating‘self-regenerating’ suppressor c’ suppressor cartridge incorporating anartridge incorporating an electrolysis cell is also a
electrolysis cell is also available where Hvailable where H33OO++ and O and O22 are continually formed by are continually formed by
the electrolysis of a stream of deionized water passing through an anode the electrolysis of a stream of deionized water passing through an anode compartment and similarly, OH¯
compartment and similarly, OH¯ and Hand H22 are formed in a cathode compartment. are formed in a cathode compartment.
Both compartments are separated from the eluent either by cation or Both compartments are separated from the eluent either by cation or anion-exchange membranes depending on whether anionic or cationic analytes are exchange membranes depending on whether anionic or cationic analytes are toto be separated.
be separated. vi)
vi) Chiral ChromatographyChiral Chromatography
Quite often only one enantiomer possesses the
Quite often only one enantiomer possesses the desired therapeutic activitydesired therapeutic activity whereas the other may be inactive or even harmful. The separation of whereas the other may be inactive or even harmful. The separation of enantiomers by HPLC using chiral stationary phase (CSP) is based on the enantiomers by HPLC using chiral stationary phase (CSP) is based on the formation of
formation of transient diastereoisomtransient diastereoisomeric compounds beric compounds between theetween the
Electric Electric in intete raratortor
enantiomorphs of the solute and the chiral selector which is an integral part of enantiomorphs of the solute and the chiral selector which is an integral part of the stationary phase. The difference in stability between these complexes results the stationary phase. The difference in stability between these complexes results in difference in their retention times, the
in difference in their retention times, the enantiomer forming the less stableenantiomer forming the less stable complex being eluted first.
complex being eluted first.
A large number of chiral phases are commercially available. All of
A large number of chiral phases are commercially available. All of these arethese are coated on silica gel support. The coating itself is
coated on silica gel support. The coating itself is a polymeric material to whicha polymeric material to which an optically active isomer is bonded. For example, the
an optically active isomer is bonded. For example, the ll form of the amino acid, form of the amino acid, proline has been bonded to
polystyrene-proline has been bonded to polystyrene- p p-divinylbenzen-divinylbenzene, a e, a cross linkedcross linked copolymer to give an optically active stationary phase for the separation of copolymer to give an optically active stationary phase for the separation of racemic mixtures of amino acids. In this case,
racemic mixtures of amino acids. In this case, CuCu2+2+ ions are introduced into the ions are introduced into the solution of the analyte
solution of the analyte enantiomers to be separated whereby a tenantiomers to be separated whereby a t ernary complex,ernary complex, as shown in Fig. 8.11. is formed between the stationary phase, amino acid anion as shown in Fig. 8.11. is formed between the stationary phase, amino acid anion and Cu
and Cu2+2+. . The formation constant for The formation constant for this complex differs forthis complex differs for d d and and ll forms of forms of the analyte amino acid; thus, making their separation possible.
the analyte amino acid; thus, making their separation possible.
Fig. 8.11: Illustration of a t
Fig. 8.11: Illustration of a t ernary complex formed between an L-proline bonded phase,ernary complex formed between an L-proline bonded phase, an analyte amino acid and a Cu
an analyte amino acid and a Cu2+2+ ion ion
Cyclodextrin-b
Cyclodextrin-bonded stationary phases have onded stationary phases have been demonstrated to bbeen demonstrated to bee particularly efficient in resolving structural isomers. Some examples particularly efficient in resolving structural isomers. Some examples are- are-prostaglandin A
prostaglandin A11, A, A22and Band B11BB22,, αα- and- and β β -naphthols,-naphthols, oo,,oo′′ and and p p,, p p′′-biphenyls and-biphenyls and
the
the orthoortho-,-, metameta- and- and para para- isomers of nitrophenol, nitroaniline, xylene, cresol- isomers of nitrophenol, nitroaniline, xylene, cresol and aminobenzoic acid.
and aminobenzoic acid.
Recently introduced graphitized carbon and new generation of rigid porous Recently introduced graphitized carbon and new generation of rigid porous polymeric microbeads based on styrene/divinyl benzene as alternatives to polymeric microbeads based on styrene/divinyl benzene as alternatives to silicasilica can be used over a wide range of pH between 1
can be used over a wide range of pH between 1 to 13. Some examples of columnto 13. Some examples of column packings used in HPLC and their applications are listed in
packings used in HPLC and their applications are listed in Table 8.5.Table 8.5. Table 8.5:
Table 8.5: Some Typical Some Typical Column Packings Column Packings Used in HPUsed in HPLCLC
Packing
Packing Mode Mode of of HPLC HPLC ApplicationsApplications Microparticulate silicas;
Microparticulate silicas; spherical or irregular spherical or irregular particles; mean particle size particles; mean particle size 3, 5 and 10µm
3, 5 and 10µm chemically modified chemically modified versions of the above versions of the above (bonded-phase packings) (bonded-phase packings)
LSC
LSC (adsorption) (adsorption) Non-polar Non-polar to to moderately moderately polarpolar mixtures,
mixtures, ee..gg., polyaromatics, fats, oils,., polyaromatics, fats, oils, mixtures of isomers
mixtures of isomers
Octadecyl (ODS or C
Octadecyl (ODS or C1818) ) BP BP (bonded (bonded phase)phase)
and
and Ion Ion PairPair Chromatography Chromatography (IPC)
(IPC)
Wide range of moderately polar Wide range of moderately polar mixtures,
mixtures, ee..gg., pharmaceuticals and., pharmaceuticals and drugs, amino acids
drugs, amino acids Octyl (C
Octyl (C88) ) BPC, BPC, IPC IPC More More polar polar mixtures,mixtures, ee..gg., pesticides,., pesticides,
herbicides, peptides, metabolites in herbicides, peptides, metabolites in body fluids
body fluids
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