EXP 35 - High Performance Liquid Chromatography

8/2/2019 EXP 35 - High Performance Liquid Chromatography

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METU CHEMICAL ENGINEERING DEPARTMENT

ChE420 CHEMICAL ENGINEERING LABORATORY III

EXP. 35

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

OBJECTIVE

The purpose of this experiment is to learn the basic principles of high performance

liquid chromatography (HPLC), which is one of the most common analysis techniques to

identify, purify and quantify the compounds of multicomponent systems. In this experiment,

you will use an Ion Exclusion type of HPLC with Ultraviolet Spectrophotometry Detector to

separate and analyze the constituents of organic solutions.

GENERAL CONCEPTS IN CHROMATOGRAPHY

Chromatography is essentially a physical method of separation in which the

components to be separated are distributed between two phases, one of which is stationary

(stationary phase) while the other (the mobile phase) moves in a definite direction [1]. The

chromatographic separations therefore have three distinct features: a) they are physical

methods of separation; b) two distinct phases are involved; c) separation results from

differences in the distribution constants of the individual sample components between the two

phases. Chromatographic separations depend on a favorable contribution from

thermodynamic and kinetic properties of the compounds to be separated. Molecular

interactions leading to differential affinities of solutes for two phases are associated with polar

forces (dipole-dipole and H-bonding) and dispersion forces (induced dipole) [2]. Useful

chromatographic separations require an adequate difference in the strength of physical

interactions for the sample components in the two phases, combined with a favorable

contribution from system transport properties that control the movement within and between

phases. Individual compounds are distinguished by their abilities to participate in common

intermolecular interactions in the two phases, which can generally be characterized by

equilibrium constant, and is thus a property that can be predicted from chemical

thermodynamics.


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A convenient classification of chromatographic techniques can be made in terms of the

physical state of the phases employed for the separation. When the mobile phase is a gas and

the stationary phase is a solid or liquid, the separation technique is known as gas

chromatography (GC). In GC, the analyte (the chemical constituent that is of interest in the

analytical procedure) must be vaporized in order for migration through the capillary to occur.

When the mobile phase is a supercritical fluid, and the stationary phase being either a solid or

an immobilized liquid, the separation technique is called supercritical fluid chromatography

(SFC). For GC and SFC, the dominant separation mechanisms are partitioning between bulk

phases and interfacial adsorption.

Transport of solute zones in column chromatography occurs entirely in the mobile

phase. Transport is an essential mechanism of the chromatographic system since the common

arrangement for the experiment employs a sample inlet and detector at opposite ends of the

column with sample introduction and detection occurring in the mobile phase. In a typical

elution chromatography, which is the most convenient method for analysis (and also used in

this analysis), the mobile and stationary phases are normally at equilibrium. The sample is

applied to the column as a discrete band by injection, and sample components are

successively eluted from the column diluted by the mobile phase. The mobile phase must

compete with the stationary phase for the sample components, and for a separation to occur,the distribution constants for the sample components resulting from the competition must be

different.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Modern liquid chromatography is dominated by the use of inorganic oxides as

stationary phases with organic functional groups chemically bonded to their surface, known as

bonded phases, and to a lesser extent, porous polymers. High-performance liquid

chromatography (HPLC) is unquestionably the most widely used of all of the analytical

separation techniques due to its sensitivity, its ready adaptability to accurate quantitative

determinations and its suitability for separating nonvolatile species or thermally fragile ones

[1-3]. About an order of magnitude smaller particle sizes with respect to open column LC,

allow HPLC to attain higher column efficiencies, while higher pumping pressures are required

to overcome the large back-pressures due to the smaller packing particles. HPLC has awidespread applicability to substances that are of prime interest to industry, to many fields of


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science, and to the public. Examples of such materials include amino acids, proteins, nucleic

acids (DNA and RNA), hydrocarbons, carbohydrates, drugs, terpenoids, pesticides,

antibiotics, steroids, metal-organic species, and a variety of inorganic substances.

Types of Liquid Chromatography

Liquid Chromatographic modes can be categorized in five different groups according

to their retention mechanism as can be seen in Figure 1:

Figure 1: Liquid Chromatography Modes

The mechanism of interaction between the analyte molecules and the stationary phase

particles can be categorized as (a) adsorption interactions, (b) partitioning, and (c) ion-

exchange. When the stationary phase is a solid, and interfacial adsorption is the dominant

separation mechanism, the technique is referred to as liquid-solid chromatography (LSC).

Polar silica, alumina or metal oxide particles are used as the stationary phase, while the

solvent systems used are typically mixtures of relatively nonpolar liquids. The combination of

a polar stationary phase and a non-polar mobile phase is called normal-phase HPLC, where

the analyte is retained due to the interactions between its polar functional groups and the polar

groups on the packing surface. Analytes elute in the order of increasing polarity, i.e. the least

polar compound will elute from the column first, followed by each compound in the order of

increasing polarity. This technique is preferred to separate analytes with low to intermediate

polarity, and sufficient solubility in nonpolar solvents.

Partition chromatography is based on solubility interactions between the analyte and

the stationary phase. In most cases, the stationary phase is a porous silica particle to which an

organic moiety (a functional chemical group) has been bonded. The organic substituent serves

effectively as a bound liquid that can interact with analyte molecules. This type of stationary

phase is called a bonded phase packing. The most popular bonded phase packings are based

Liquid Chromatography

Adsorption

Competition

between liquid

mobile phase and

solid adsorbent

Partition

Competitionbetween liquid

mobile phase and

liquid stationary

phase

Size Exclusion

Molecular sieving

Affinity

Lock and Key

mechanism

Ion Exchange

Competitionbetween liquid

mobile phase and

ionic stationary

phase


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on C8 or C18-alkyl chains. This creates a non-polar stationary phase. Solvent systems used

with these stationary phases are typically mixtures of polar liquids such as water, methanol,

and acetonitrile. The use of a non-polar stationary phase and a polar mobile phase is called

reversed-phase (RP) HPLC, which has become the most popular separation mode in

modern liquid chromatography. Since the analytes are attracted to the surface by their non-

polar functional groups, they elute (to be washed out of the column, within the mobile phase)

in the order of increasing polarity, i.e. the most polar compound will elute from the column

first, followed by each compound in the order of decreasing polarity. Reverse phase

chromatography is preferred for separating compounds with moderate to high polarity, and

sufficient solubility in polar solvents.

Ion Exchange Chromatography (IEC) employs either a porous polymeric stationary

phase or silica substrate with immobilized acidic or basic functional groups attached as ionic

sites. The dominant separation mechanism is based on electrostatic interactions between ions

in the mobile phase and those on the stationary phase, which can allow an ion exchange

reaction to take place. The magnitude of the reaction equilibrium constant represents the

affinity of an analyte ion for the stationary phase. The mobile phase is also an aqueous

solution of ionic species, which compete with the analyte for exchange sites on the stationary

phase.

Affinity chromatography (AC) is a type of liquid chromatography that makes use of

biological-like interactions for the separation and specific analyses of sample components [4].

The stationary phase is a solid with immobilized molecular recognition sites which can

establish a specific interaction with a specific solute molecule; for example, the immobilized

molecule may be an antibody to some specific protein. In this specific example, when a solute

containing a mixture of proteins is passed by this molecule, only this specific protein can react

with this antibody, and so binds to the stationary phase. This protein can later be extracted by

changing the ionic strength or pH of the mobile phase.

If the stationary phase is a solid with a controlled pore size distribution, and solutes are

separated by size differences, i.e. molecular sieving, then the technique is called size-

exclusion chromatography (SEC). On the contrary to the previous chromatography modes

explained above, this type of chromatography which is also known as gel permeation or gel

filtration, lacks an attractive interaction between the stationary phase and the solute. Since the

pores are small and exclude the larger solute molecules, only the smaller molecules can enter


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the gel and thus flow through a larger volume. Therefore, larger molecules have less retention

times, passing through the column at a faster rate than the smaller ones.

Instrumentation for HPLC

The basic components of a high-performance liquid chromatographic system is shown

in Figure 2. The instrument consists of (a) eluent containers for the mobile phase, (b) a pump

to move the eluent and sample through the system, (c) an injection device to allow sample

introduction, (d) a column(s) to provide solute separation, (e) a detector to visualize the

separated components, (f) a waste container for the used solvent, and finally (g) a data

collection device to assist in interpretation and storage of results [3].

The pump, injector, column, and detector are connected with tubing of narrow innerdiameter. The choice of detector is based on intrinsic properties of the solute. Often more than

one detector can be used to maximize sample information and conform peak identities.

Figure 2. Schematic diagram of the basic modules of an HPLC system.

The pumping system with reciprocating pistons can supply either an isocratic or a

gradient elution depending on the number of pumps used in the system. In isocratic elution,

the mobile phase composition is kept constant throughout the analysis, while in gradient

pumping systems, more than one solvent can be used, and so the composition of the mobile

phase can be altered, which requires multiple pumps. The sample injection of the system can

be either a manual injection using 6-port valve, or an automated sample injection device. Two


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types of detectors are widely used for monitoring separations. An absorbance detector

measures the absorbance of a particular wavelength of ultraviolet light, usually tuned for

aromatic compounds. The alternative is a detector that monitors changes in the refractive

index of the solvents being eluted. This allows for detection of compounds that may be

transparent to UV radiation.

One of the most critical factors in HPLC analysis is the quality of the solvents used

[3]. In addition to being particulate and contaminant free, solvents must contain minimal

levels of dissolved gases. Dissolved gases come out of solution when the eluents are pumped

through the system, and the probability of gases bubbling out of solution increases when two

or more liquids are mixed. The microbubbles frequently become attached to check valves, and

interfere with accurate flow. In addition, they can become lodged in detector cells, creating

baseline instability. Therefore, the mobile phase and all the solvents must be degassed prior to

their use in the instrument.

The Output of Chromatography-Chromatograms

The information obtained from a chromatographic experiment is contained in the

chromatogram [1]. When the elution mode is used, this consists of a plot of a detector

response (y-axis) as a continuous function of time or volume of mobile phase passed through

the column (x-axis). The chromatogram contains a number of peaks of various sizes rising

from a baseline. Information readily extracted from the chromatogram includes an

identification of a samples complexity from the number of observed peaks; qualitative

identification of sample components from the accurate determination of the peak positions;

quantitative assessment of the relative concentration or amount of each component from their

peak areas; and the characteristic physical properties of either the solute or the

chromatographic system from the peak positions and profiles. The position of a peak in a

chromatogram is characterized by its retention time (tR), which is the time taken for a specific

solute to reach the detector, or retention volume (V R), which is the volume of solution that is

pumped through the detector before a specific peak is eluted (Figure 3).

There are four major concepts, in terms of which chromatography is described:

capacity, efficiency, selectivity and resolution [3]. The capacity and selectivity of the column

are variables that are controlled largely by the column manufacturer, whereas efficiency and

resolution can be controlled to some extent by the operator. To obtain the best possibleseparation, the efficiency of the chromatographic system must be optimized in order to


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minimize band broadening. The column should have the capacity to retain the solutes, and the

ability to separate sample components efficiently. The capacity factor kR of a column is a

direct measure of the strength of the interaction of the sample with the packing material:

= 0

0Eqn 1

where tR is the retention time and t0 is the time taken for non-retained species to reach the

detector (hold-up time).

Figure 3: Chromatographic terms used to calculate column capacity

The capacity factor of a column is mostly a function of the packing material, but can

be manipulated to a degree by varying the solvent strength. The higher the kR, the greater is

the ability of the column to retain solutes. However, this also means longer analysis times;

therefore, a compromise between resolution and analysis time must be reached. A kR value

between 2 and 5 represents a good balance between the analysis time and resolution.

The selectivity () of chromatographic separation is a measure of the difference in

retention times between two given peaks and describes how effectively the system can

separate two compounds.

=0

0=

Eqn 2

The selectivity of a column is primarily a function of the packing material; although

the chromatographer has some control using the mobile phase or temperature. An effective

way to increase is to change the composition of the mobile phase.

The resolution represents the degree of separation between neighboring solute peaks.

It depends on the selectivity (), efficiency (N), and capacity (kR) of the column.


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=1

4

1

(1 2 )

1+Eqn 3

Peak broadening occurs generally due to sample components interactions with the

stationary phase, and the torturous path that the solute has to take through the packingmaterial. The efficiency of a column, N, is a number that describes peak broadening as a

function of retention, and can be described simply in terms of the number of theoretical plates.

In plate theory, the column is considered to consist of a number of thin plates, each of which

allows a solute to equilibriate between the stationary and mobile phases. The greater the

number of theoretical plates, i.e., the efficiency, N, the more efficient the column is

considered to be. The movement of a solute along the column is viewed as a stepwise transfer

from one theoretical plate to the next. N can be defined using the half-height method as:

= 5.545(

/)2

where W1/2 is the peak width at the half height of the peak (in units of time) (Figure 4).

Figure 4: Baseline width (Wb) and width-at-half-height (W1/2) of a chromatographic peak.

The efficiency can be varied by changing the physical parameters such as length,

diameter, and construction material of the container of the column. It can also be varied by


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changing chemical parameters such as the size of the particles constituting the packing

material or the mobile phase velocity.

Note: The efficiency of a column is described more realistically with the rate theory which

avoids the assumption of an instantaneous equilibrium, and addresses the diffusional factors

contributing to band broadening in the column, such as eddy diffusion, longitudinal diffusion,

and resistance to mass transfer in the stationary phase and the mobile phase. Contribution of

these factors to efficiency is represented in a Van Deemter equation, and a plot is used to

determine the experimental conditions required to obtain the most efficient system [3].

References:

1- C. F. Poole, The Essence of Chromatography, 1st

ed., Elsevier, Amsterdam, 2003.2- D. A. Skoog, J. J. Leary, Principles of Instrumental Analysis, 4th ed., Saunders College

Publishing, Fort Worth, 1992.

3- A. Weston,P. R. Brown,HPLC and CE: Principles and Practice, 1st ed., AcademicPress, San Diego,1997.

4- D. S. Hage, Affinity Chromatography: A Review of Clinical Applications, ClinicalChemistry, 45, 5, 593-615, 1999.

PRELIMINARY WORK

Be prepared to answer the following questions prior to the laboratory session.

- What is HPLC used for? What is the main difference between an HPLC and GC?- What is the basic principle of HPLC?- What are the types of liquid chromatography? What are the separation mechanisms in

each type?

- What are the most commonly used detectors in HPLC? Get familiarize with theirworking principles.

- What is the principle of ion exclusion chromatography? (Please use instrumentalanalysis textbooks or other sources to learn the answer).

- What are the basic working priciples of reciprocating piston pumps and six-portvalves?

- Study the acid dissociation phenomena, look into the pK a values of the commonstrong and weak acids.
http://www.google.com.tr/search?hl=tr&tbo=p&tbm=bks&q=inauthor:%22Andrea+Weston%22http://www.google.com.tr/search?hl=tr&tbo=p&tbm=bks&q=inauthor:%22Phyllis+R.+Brown%22http://www.clinchem.org/search?author1=David+S.+Hage&sortspec=date&submit=Submithttp://www.clinchem.org/search?author1=David+S.+Hage&sortspec=date&submit=Submithttp://www.google.com.tr/search?hl=tr&tbo=p&tbm=bks&q=inauthor:%22Phyllis+R.+Brown%22http://www.google.com.tr/search?hl=tr&tbo=p&tbm=bks&q=inauthor:%22Andrea+Weston%22


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- Describe the following chromatographic parameters: column capacity factor,selectivity, resolution and number of theoretical plates. How would you manipulate

these parameters in a chromatographic system?

EXPERIMENTAL PROCEDURE

1. Make sure that a baseline has been attained before starting to analysis.2. Take readings of the mobile phase flow rate, column temperature, minimum and

maximum pressure limits of the pumps, elution profile, sample volume that is

injected, composition of the mobile phase.

3. Preparation of the mobile phase; (this will have been already prepared for therun, so you can skip this step; but you must know how it is prepared.)

Prepare 8 mM of sulfuric acid solution with Millipore Q (high purity water). Put

435 l of 96-98% (w/w) sulfuric acid (as received from Merck, 5 L bottles) into 1

L of MilliQ. Filter it through 0.45 m cellulose acetate membrane filter. Degass the

solution for 10-15 minutes at room temperature. Filtered and degassed mobile

phase is now ready to use in HPLC. The mobile phase can be used at most 1 week;

for the next week, the fresh solution should be prepared since microbial growth

will occur in the solution and it will obviously absorb air with time.

4. To obtain the calibration curves for the standards;The standards, oxalic acid, citric acid and malic acid will be prepared at a

concentration of 500 ppm (stock solutions). For standard preparation, mobile

phase solution should be used. Prepare different standards, each with one type of

acid solution at a certain concentration less than 300 ppm. These standards will

allow you to detect the retention times of each acid. Next, prepare three different

standards, each with all three acids at a certain concentration again less than 300

ppm, and different than the concentration of the first set of standards. Use a glass

pipette (1ml) and sample vials for the preparation of these standards.


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5. Press single injection and upload the method file (recently uploaded in thesoftware). Also upload a data file which represents the name of the single sample

that will be analyzed in the device. Fill the sample volume as 20 L and press OK.

A small box is going to appear which specifies that the device waits for the

manual injection.

You will not be using an auto-sampler so you have to perform injections manually

through the injection port and wait until each analysis will be completed. Before

each injection make sure that you rinse your syringe (stainless steel syringe) with

mobile phase to remove any contaminants. Slowly and completely fill the syringe

with your sample. Before injection, make sure that the sample in the syringe does

not contain any bubbles. Any air bubble can cause damage for the device. These

syringes are non-dispensable and expensive. So please pay attention while using

it.

Before injection, the injection port should be in load mode (left). Put the syringe

to the injection port gently. Make the injection. After you make the injection of 50

l and remove the injector from the port, you will turn the port to the inject mode

(right), at the same time you will see the running alert on the monitor.

6. After you complete this procedure for all of your standards, repeat the sameprocedure for the samples with unknown compositions.

7. Obtain your runs data report. Make sure you have all the data you need tocomplete the post-run calculations for your report.

DATA SHEET

Initial readings of the main menu

Analysis Time:

Flow rate of mobile phase:

Column temperature:

Pressure limits of the pump:

UV absorbance wavelength:

Injection volume:

Composition of the mobile phase:


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STANDARDS CompoundConcentration

(ppm)Retention

timePeak Area

1

2

3

4All threeacids

5All threeacids

6All threeacids

UNKNOWN

Non-retained

species,Rt

Non-retained

species,Area

Rt, 1 Area1 Rt, 2 Area2 Rt, 3 Area3

wh1 wh2 wh3

CALCULATIONS

1. Construct the calibration curve for each species. Use least squares procedures tocompute the slope, y-intercept, r2, and standard error of estimate.

2. Determine the concentration of the components in the unknown solution.3. Choose one of your unknown samples and calculate:

a. Capacity factor of the column for each species


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b. Selectivity of the column; i.e., selectivity for citric acid over oxalic acid, andfor malic acid over citric acid

c. resolution of the two sets of neighboring peaksd. efficiency of the column for each species: i.e., the number of theoretical

plates for each species

Please discuss the results of your calculations. Your discussions should include your

comments about the efficiency and reliability of this analysis, and if you have any suggestions

to improve it.

EXP 35 - High Performance Liquid Chromatography

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Transcript of EXP 35 - High Performance Liquid Chromatography