HPLC Lab course

Grundlagen der Chromatographie und chromatographischen Methoden VAK 02-03-5-AnC2-2

Introductory experiments in high performance liquid chromatography

January 29, 2008

Equipment
HPLC system consisting of a pump (0.5 to 2 mL/min) with pressure display, a 6-port injection valve with e.g. 20 L sample loop, a reversed phase column, e.g. 250 mm, 4.6 mm i.d. packed with octadecylsilica with a particle size of 7 m a UV detector with a detection wavelength of 254 nm an integrator, a 25 L glass syringe with blunt needle point

Substances
Deionized water Methanol Acetonitril Triethylammonium formate Thiourea solution 3 References of aromatics Mix of aromatics 7 Ref. of cAMP and metabolites Mix of cAMP and its metabolites from the WEK (water deionzied cold) tap in the lab HPLC grade HPLC grade 1 M solution in water 50 mg/L in water Solutions of phenol, anisol, and toluene in methanol Methanolic solution containing phenol, anisol, and toluene Aqueous solutions of cAMP, 5-AMP, adenosine, inosine, hypoxanthine, and xanthine Aqueous solution containing all seven compounds

Preparation of eluents

The eluents used in the next experiment are listed in Table ??, together with the amounts that will presumably be used. Please measure the volumes of water and methanol separately and combine them afterwards, in order to get a clearly dened mixture.

Table 1: Eluent compositions to prepare Eluent A B C D Composition 100 %MeOH 85 %MeOH / 15 % H2 O v/v 75 %MeOH / 25 % H2 O v/v 65 %MeOH / 35 % H2 O v/v Amount 500 mL 250 mL 250 mL 250 mL

HPLC Lab course

Grundlagen der Chromatographie und chromatographischen Methoden VAK 02-03-5-AnC2-2

Questions
1. Prior to the experiment any solvent eluent used must be degassed on the water pump in an ultrasonic bath. Why is this necessary? 2. By watching the degassing procedure, the difference between degassing and boiling should become evident. Why should boiling be avoided in this process?

Operating the HPLC system

Before starting, you should thoroughly familiarize yourself with the HPLC, and its individual components and their function. Make sure that the solvent lter is covered with solvent at all times, and that the waste container is not full. This will prevent undesirable ooding (also in regard to possible health damages caused by organic fumes). When eluent A is connected, the pump is preliminarily set to a ow rate of 0.5 mL/min and after a few minutes the ow rate can be elevated to 2.0 mL/min. Does the actual ow rate deviate from the nominal value (set value)? Note the pressure at various ow rates (2.0; 1.5; 1.0; 0.5 mL/min) starting with the lowest value (0.5 mL/min). Apart from the solvent supply, the pressure display should be watched. When using pure methanol at a ow rate of 1.5 mL/min the pressure will be about 70 bar, depending on the length of the colum; with other solvents, the pressure will be higher. The tolerable maximum for the pumps in use is around (200 bar). Too high a pressure will indicate that the lter in the column assembly is plugged (inform supervisor!). When there are strong uctuations in pressure (mostly accompanied by irregular pump noises), there is air in the device. In this case the pressure valve must be opened immediately while pumping is continued, in order to remove the air. Moreover, one should look for leaks, which will (in the extreme case) result in eluent dripping down from the leaking equipment. Then the detector is switched on and the system is equilibrated at a ow rate of 1.5 mL/min. Make sure that the base line is stable and conrm the detection wavelength of you detector to be 254 nm.

Reporting the results

Plot pressure vs. ow rate

Questions
1. What is the relation between pressure and ow rate?

Determination of dead time and dead volume

For use in HPLC, only syringes with a blunt needle are employed. Please do not force the piston (pistons which are not easily movable indicate contamination) but move it preferably with two ngers and pull it up the same way. When applying pressure on the piston from above it will break or be kinked, rendering it unusable as another piston will not t in the syringe. After using the syringes ush them several times with a suitable solvent, and eventually with methanol. In order to determine dead time and dead volume, a thiourea solution is injected at different ow rates and different concentrations. Use eluent A (pure methanol) for these experiments.

Questions
1. How long is the retention time (dead time)? 2. How large is the dead volume? 3. Is the dead time and/or the dead volume dependent on the ow rate? 4. Is the dead time and/or the dead volume dependent on the quantity of thiourea solution injected?

HPLC Lab course

Grundlagen der Chromatographie und chromatographischen Methoden VAK 02-03-5-AnC2-2

5. Is the dead time and/or the dead volume dependent on the thiourea concentration in the sample? 6. What are the requirements which a compound must satisfy in order to enable the determination of dead time/dead volume?

Determination of column data

A test mixture consisting of toluene, anisole, and phenol is injected into the HPLC using eluents A, B, C, and D (take care to switch off the pump each time you change solvent, otherwise you risk pumping air into the column). The ow rate in each case is set to 1.5 mL/min. Identify the peaks by comparing retention times and check by injecting individual substances. After the measurements, the whole system must be ushed for 15 to 20 minutes with pure methanol. During the rinsing, pressure and absorbance have to be watched.

Reporting the results

Tabulate the following values for the chromatograms obtained with all four eluents for each individual compound: capacity factor k, number of theoretical plates N , and height of a theoretical plate H For each eluent, calculate the resolution R between anisol and toluene. Plot the decadic logarithm of the capacity factor log k versus the volume fraction of methanol in the mobile phase for all three substances in the same graph. Is a linear relationship evident? If so, determine coefcients S and kw according to the basic equation of the linear solvent strength model log k = log kw S

Questions
1. What is the interpretation of kw and S? Which properties of the individual compounds do they relate to? 2. What happened if the lines for two compounds intersect?

Optimizing the separation of cAMP metabolites

Cyclic adenosine-3,5-monophosphate (cAMP) is metabolized (e.g. in bovine liver) via adenosine-5-monophosphate (5-AMP), adenosine, inosine, hypoxanthine and xanthine to yield uric acid (Figure ??) Get aquainted with the structures of these metabolites and the type of reaction and the enzymes involved. Before starting the separation experiments it is required to color the molecules in accordance with Prof. Jastorffs color code and to associate the given pKa values with the functional groups of every molecule. Only based on this theoretical basis you can think about the inuence of different buffered solvent eluents wich is important for an effective and straightforward design of the separation experiments. The aim is to optimize the separation conditions to a degree that a quantitative determination of all metabolites can be achieved. This may not be possible. Optimization shall be started with a 7 % acetonitrile solution in water containing 2 mM of TEAF and a pH of 6.5. Only change one parameter at a time! When all measurements are taken, ush the system for 15 to 20 minutes with a methanol/water mixture.

Questions
1. Can you think of other ways to improve the separation? 2. Does the order of peaks agree with your expectations based on your understanding of the molecular interactions?

HPLC Lab course

Grundlagen der Chromatographie und chromatographischen Methoden VAK 02-03-5-AnC2-2

NH2 N N N

NH2 N

N O O
-

cyclic nucleotide phosphodiesterase

O

O
-

N O O
-

P O

O O

P O

3',5'-cAMP pKa: <2; 3,8

OH

5'-AMP pKa: 0,7; 3,5; 6,7

OH

OH

NH2 N HN

O N

5'-nucleotidase
HO

adenosine deaminase
HO

adenosine pKa: 3,5; 12

O

OH

OH

inosine

OH

OH

pKa: 1,2; 8,75; 12,33

O

nucleoside phosphorylase
HN N

xanthine oxidase
O HN N

xanthine oxidase

NH

N H

NH

hypoxanthine pKa: 1,98; 8,94; 12,10

O H N O O N H NH H3C H3C

xanthine pKa: 0,8; 7,44; 11,12

H3C O N
+

HN

triethylammonium ion

formate ion

uric acid pKa: 5,4; 10,30

source of pKa values: Biochemist's Handbook

Figure 1: Structures of cAMP metabolites together with their dissociation constants and structures of the ions in the ion pairing buffer.