High Pressure Liquid Chromatography Laboratory Training Guide

Keaton Smith, last update Fall 2011

Principles and Applications

During the past three decades, an analytical method has been developed that now surpasses traditional liquid chromatographic techniques in importance for analytical separations. This technique, high pressure liquid chromatography (HPLC), is ideally suited for the separation and identification of amino acids, carbohydrates, lipids, nucleic acids, proteins, pigments, steroids, pharmaceuticals, and many other biologically active molecules.

The future promise of HPLC is indicated by its classification as “modern liquid chromatography” when compared to other forms of liquid chromatography such as paper, thin-layer or column chromatography, which now referred to as “classical” or “traditional.” Compared to the classical forms, HPLC has several advantages:

1. Resolution and speed of analysis far exceed the classical methods.2. HPLC columns can be reused without repacking or regeneration.3. Reproducibility is greatly improved because the parameters affecting the efficiency of the

separation can be closely controlled.4. Instrument operation and data analysis are easily automated.5. HPLC is adaptable to very small sample sizes or large-scale, preparative procedures.

These advantages have made HPLC the fastest growing separation technique in chemistry, but its use in undergraduate laboratories is limited because of the high cost of the instruments, columns and high-purity solvents required (e.g. HPLC-grade water costs about $45 per liter).

As in column chromatography, the mobile phase or solvent is a liquid that carries the sample through a column packed with fine particles that interact with the sample components to different extents, causing them to separate. The sample is injected into the column and detected as it leaves the column. The samples passage through the column is recorded as a series of peaks on a chromatogram. In HPLC, the stationary phase stays in place inside the column, and is commonly a viscous liquid chemically bonded to the column or particles in the column. Each component of the sample is partitioned between the liquid mobile phase and liquid stationary phase according to a ratio--the partition coefficient--that depends on its solubility in each liquid. The components of a mixture generally have different partition coefficients in the liquid phases, so they pass down the column at different rates. Retention time of an analyte in HPLC is defined as the time necessary for maximum elution of a particular analyte. Resolution indicates how well analytes are separated, i.e. column efficiency. HPLC has an improved efficiency over column chromatography due to its reduced particle size.

Most column chromatography packings (stationary phase) contain particles (e.g. organic coated silica beads) with diameters in the 75 - 175 µm range, while most modern HPLC packings have particle diameters in the 3 - 10 µm range which increases the separation efficiency dramatically. However such a small particle size inhibits the flow rate; therefore, HPLC solvent flow is obtained by applying a pressure differential across the column. A powerful pump is utilized to force eluents (the entire solution entering the column) through the column at pressures up to 6000 psi or 41 bar. A combination of high pressure and adsorbents of small particles size leads to the high resolving power and short analysis times characteristics of HPLC.

Some HPLC columns contain only solid adsorbent such as silica gel or alumina. Such a stationary phase is more polar than the eluent, so nonpolar components are eluted faster than polar ones. This is an example of normal-phase chromatography, where a polar stationary phase and a less polar solvent, or mobile phase, is used. Most modern HPLC columns contain a bonded liquid phase–an organic phase that is chemically bonded to coat particles of silica gel. Silica gel contains silanol (—Si—OH) groups to which long hydrocarbon chains, such as octadecyl (—(CH2)17CH3) groups (indicated as C18), can be attached. This is an example of reversed-phase chromatography where a nonpolar (or weakly polar) stationary phase and more polar solvent is used. The bonded stationary phase in this example is less polar than the eluent, which may be a mixture of water with another solvent such as methanol, acetonitrile, or tetrahydrofuran. Thus polar components will spend more time in the eluent than in the stationary phase and will be eluted faster than nonpolar ones, reversing the usual order of elution. Some of the most popular reverse-phase packings consist of silica gel with bonded methly (CH 3), phenyl (—C6H5), octyl (—(CH2)7CH3), octadecyl (—(CH2)17CH3), cyanopropyl (—(CH2)3CN), and aminopropyl (—(CH2)3NH2) groups.

Other stationary phases operate by still different mechanisms. The stationary phase for size-exclusion chromatography is a porous solid that separates molecules based on their effective size and shape. Small molecules enter the narrowest openings in the porous structure, while larger molecules find fewer openings to enter, and still larger molecules may be completely excluded from the solid phase. Thus large, bulky molecules pass down the column faster than smaller molecules. A stationary phase for ion-exchange chromatography has ionizable functional groups that carry a negative or positive charge over a suitable pH range, attracting ionic solutes from solution. Such stationary phases are mainly used to separate ionizable organic compounds such as carboxylic acids, amines, and amino acids. Chiral stationary phases that can separate enantiomers and determine their optical purity are also available. By utilizing variations in stationary and mobile phases, many classes of compounds may be quantified.

There are two general method of solvent delivery in HPLC. Isocratic elution is performed with a single solvent (or constant solvent mixture). If one solvent does not provide sufficiently rapid elution of all components, then gradient elution can be used, where increasing amounts of solvent B is added to solvent A to create a continuous gradient. In gradient elution the column requires 10-20 empty column volumes of initial solvent in order to reequilibrate the stationary phase with the solvent before the next sample is introduced.

Very pure and expensive HPLC-grade mobile phase solvents are required to avoid degrading costly columns with impurities and to minimize detector background signals from contaminants. A filter is used on the intake tubing in the solvent reservoir to reject micron-size particles. Sample and solvent can be passed through a short, expendable guard column which contains the same stationary phase as the analytical column and collects strongly adsorbed species. All eluents (i.e. the mobile phase, sample and any other liquids entering the column) should be degasses, which may be done by various methods including refluxing, vacuum filtration, ultrasonic vibration, and inert gas purging. Air bubbles create difficulties with pumps, columns, and detectors. Vacuum filtration should be performed on liquids to remove particulates that could be drawn into the pump which could cause a blockage in the column.

Depending on the chemical(s) desired to be measured there are sequences described in various sources concerning how to choose the best HPLC method, including how to determine what type of column/stationary phase, mobile phase and detector should be used.

Instrumentation

The basic components of an HPLC system are diagrammed in Figure 1. The solvent reservoir should have a capacity that allows for the complete analytical procedure, usually larger than 500 ml. The pumping

system provides a constant, reproducible flow of mobile phase solvent through the column. A constant-volume pump is commonly used as it maintains a relatively accurate flow rate and produces a more precise analysis as compared to a constant-pressure pump. The syringe injector introduces the sample to the column in an efficient and reproducible manner. The column in HPLC is prepared from stainless steel or glass-Teflon tubing. Typical column inside diameters are from 2.1 to 4.5 mm and column length can range from 5 to 100 cm. Common detectors used in HPLC include differential refractometer, photometric and fluorescence detectors. Photometric detectors measure the extent of absorption of ultraviolet or visible radiation by a sample. UV detection allows most biochemicals to be detected, including proteins, nucleic acids, pigments, vitamins, some steroids, and aromatic amino acids. Aliphatic amino acids, carbohydrates, lipids, and other biochemicals that do not absorb UV can be detected by chemical derivatization with UV absorbing functional groups. UV detectors have many positive characteristics, including high sensitivity, small sample volumes, linearity over wide concentration ranges, nondestructiveness to sample, and suitability for gradient elution. Eluent collection can be performed using HPLC because it is nondestructive to the sample. Most HPLC instruments are integrated with a computer system to allow for automated control of the pump, injector, detector, and data system. Data gathering and analysis are both commonly done by computer software. As each component of a sample leaves the column, the detector responds to some property of the component and sends an electrical signal to a computer, which traces the component’s peak. The resulting chromatogram is a graph of some property of the components, commonly absorbance, plotted against time or volume.

Figure 1: A schematic diagram of a HPLC system

Example Experimental Protocol

Adenosine is a purine nucleoside, or glycosylamine, composed of adenine and a ribose moiety by a β-N9-glycosidic bond. It is commonly found attached to one or more phosphate groups creating a nucleotide, a DNA structural unit; e.g. adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP). There are only two conformations of adenosine with respect to the attached ribose units are sterically permitted, anti or syn. Coenzyme A (CoA), which functions in acyl group transfer reactions, contains adenosine as a cofactor. Nicotinamide adenine dinucleotide (NAD+), which functions in hydride transfers, and flavin adenine dinucleotide (FAD), which functions in electron transfers, both contain adenosine as cofactors. One of the most common signaling second messenger nucleotides is adenosine 3’,5’-cyclic monophosphate (cyclic AMP, or cAMP) formed from ATP in a reaction catalyzed by adenylyl cyclase. Because of adenosine’s vast involvement in biochemical processes, it has been proposed that adenosine is a viable wound healing factor. For this reason a method was formed using HPLC in order to utilize its instrumental advantages towards determining unknown concentrations of adenosine in fluid samples taken from various in vitro and in vivo studies.

The following experiment will require that you use previous knowledge of solution chemistry to create a calibration curve which will relate adenosine UV absorbance to known concentrations of adenosine by linear regression, all preformed using the HPLC method information described below. Then, samples of unknown adenosine concentration will be measured using the identical method and standard calibration curve comparison. Note that because this experiment requires instrumentation under very high pressure, a simple operation error could cause an instrumentation malfunction; therefore an instructor will be in the lab while the experiment is performed.

1) Create enough the mobile phase known described below to establish column equilibrium, run a calibration curve, and test multiple samples.

2) Create calibration curve standards by serial dilution across the expected range of detection, i.e. the range should at least span the unknown’s expected concentration.

3) Open the Varian Galaxie software, turn on the power to all of parts of the HPLC instrumentation and ensure that the software has established communication with the instruments.

4) Use the software package to (a) “wash” the injector/autosampler, (b) turn on the detector’s UV and visible lamps and (c) begin pumping the mobile phase through the instrumentation.

5) Use the software package to create a “new method” for controlling the calibration curve sequence.

6) Create a “new sequence” linked to the method that will run the calibration curve.7) Run the calibration curve and check the software package’s linear regression analysis of the data

points.8) Alter the previously created method linked to the calibration curve to enable running the

unknowns.9) Create another new sequence linked to the altered method to run the unknowns.10) After the sequence has completed create a “new report” and export all relevant data to a pdf

document.

Warning: Never close the prime/purge valve while priming or purging the pumping/solvent delivery system. This can cause a severe pressure spike that could cause leaks in the solvent flow path.

Solution and Instrumentation Requirements for Measuring Adenosine

Varian Microsorb-MV 100-5 C18 Column 0.2 µm vacuum filtered mobile phase

o 0.01 M dipotassium hydrogen phosphateo 10 % methanolo Adjust to pH 4 using phosphoric acid and sodium hydroxide

For solution chemistry, adenosine is insoluble in water or PBS at greater than 8 g/L HPLC limit of quantitation for Adenosine is estimated to be at 2 µg/ml Adenosine absorbs UV at approximately 260nm without any chemical modification Use a flow rate of 1 ml/min The retention time of adenosine is approximately 14 minutes

Sources

Boyer, Rodney. Biochemistry Laboratory: Modern Theory and Techniques. San Francisco: Benjamin Cummings, 2006.

Harris DC. Quantitative Chemical Analysis. 6th ed. New York: W. H. Freeman and Co., 2003.

Lehman, John W. Operational Organic Chemistry: A Problem-Solving Approach to the Laboratory Course. 3rd ed. Upper Saddle River, NJ: Prentice Hall, 2002.