Waters Symposium: High-Performance Liquid Chromatography
high-performance liquid chromatography: laboratory
HPLC for Undergraduate Introductory Laboratories Scott A. Van Arman and Marcus W. Thomsen Department of Chemistry, Franklin and Marshall College, Lancaster, PA 17604 Instrumental analysis continues to grow in importance in the practice of all subdisciplines of experimental chemistry. Work done in the undergraduate laboratory should parallel this trend. Many chemistry curricula are designed with a specific course for this purpose, but in our department we incorporate many modern instrumental methods within the context of the core courses (general, organic, physical, analytical and inorganic chemistry). HPLC is one such technique that is given early treatment. Most students encounter separation science in the form of thin layer chromatography (TLC) or column chromatography as part of the organic chemistry laboratory. Nearly all commercially available laboratory manuals devote a large section to the introduction of these techniques that take advantage of differential polar affinities. We have designed a set of simple HPLC separations as an introduction to chromatography early in the organic course. Added advantages of HPLC are reduction of waste and lower expenditure for disposable materials. We also make use of HPLC’s quantitative capability, which is not readily available through the use of TLC. While a variety of systems could be used to demonstrate the technique, for instructional purposes there are often some qualifications. It is usually invaluable for students to see some relevance in the experiment; but often the instrument’s limitations are in direct conflict with this need. At least for introductory laboratories, the HPLC system available is limited to isocratic systems for which the components separated can be detected by UV absorption at 254 nm. Our initial foray into the use of this technique, designed with these goals and limitations in mind, was based on the separation of analgesics as described in this Journal (1). We introduced quantitation to the analysis of the chromatogram so that students may identify the composition of an unknown commercial analgesic tablet. The solvent system does not contain acetonitrile and is more easily disposed of than that described by Haddad et al. (1a), and a much shorter column is used. Derived from this system is an adaptation of the well-known separation of nucleosides by reversed-phase HPLC (2). Using a similar solvent system to that for analgesics, an unknown “RNA digest” (3) is quantitatively analyzed by each student in the introductory organic chemistry sequence. Experimental Procedure
acetate, 5% methanol, and 94.6 % water. The solution is appropriately filtered through a 0.45-µm Nylon-66 membrane (Alltech). The flow rate used for analgesics is 2.00 mL/min and that for nucleosides is 1.00 mL/min.
Chemicals Methanol (HPLC grade), acetic acid, triethylamine, and nucleosides were obtained from Aldrich, Milwaukee, WI. Analgesic tablets, which included Anacin®, Anacin3® , Bayer® aspirin, and Excedrin® were obtained from local retail establishments. Sample Preparation For analgesic tablet analysis, the instructor prepares standard solutions of acetaminophen, acetylsalicylic acid, caffeine, and phenacetin in the filtered eluting solvent at about 1 × 10 {4 M. The students prepare their samples for analysis by grinding the tablet with a mortar and pestle. The powder is triturated in the mortar with a small amount (10–15 mL) of the eluting solvent. The mixture is quantitatively transferred to a 250-mL volumetric flask, diluted and shaken to assure complete dissolution, and then diluted to the mark. This solution is diluted by a factor of 25 in a 25-mL volumetric flask. Each of these sample solutions (ca. 1 mL) is filtered by centrifugation in a filtration tube (0.45-µm Nylon-66 membrane, Microfilterfuge™ tubes, Rainin Instrument Company, Inc.) to remove binder before injection. Standard solutions of the nucleosides (Fig. 1) in the eluting solvent are prepared by the instructor. Unknown mixtures of solid nucleoside samples are prepared using an analytical pan balance. Mixing of the solids is effectively accomplished using a WIG-L-BUG™ amalgamator grinding apparatus (Wilmad). Approximately 5 mg of an unknown sample is provided to each student. This sample is dissolved in the eluting solvent in a 50-mL volumetric flask and diluted to the mark. The sample solution is injected directly onto the column without filtration.
NH 2
O
N HO CH 2 O N H H H H OH OH
O
HO
CH 2 O N H H H H OH OH
Cytidine
Apparatus and Operating Conditions The system (the hardware can now be purchased from Bacharach) uses a Perkin-Elmer Series 100 pump, a Perkin-Elmer TriDet detector set at the UV absorption 245 nm, and a Perkin-Elmer HS-3 C18 3.3-cm cartridge column. The output from the detector is registered on a Perkin-Elmer R-50 recorder. The solvent system employed for analgesics contains 0.4% triethylammonium acetate, 13.8% methanol, and 85.8% water. The solvent system for nucleosides contains 0.4% triethylammonium
O H 3C
NH O
Uridine
N HO CH 2 O H H H H OH OH
NH 2 N
NH N
O
Thymidine
O N
NH
HO CH 2 O N H H H H OH OH
N
NH2
N N
HO CH 2 O H H H H OH OH
Guanosine
Adenosine
Figure 1. Nucleosides used for “RNA Digest”.
Vol. 74 No. 1 January 1997 • Journal of Chemical Education
49
Waters Symposium: High-Performance Liquid Chromatography
Table 1. Retention Times (t) of Nucleosides (min) and Response Factors in Units of (mmol/mL)/Unit Peak Height at Attenuation = X Mean Peak Response Conc. X Mean t Nucleoside Height (mmol/mL) Factor (mm) Guanosine
4.04 × 10-5
16
1.60
30.9
1.31 × 10-6
Adenosine
4.12 ×
10-5
16
4.12
12.8
3.22 × 10-6
Uridine
4.08 ×
10-5
16
0.78
37.7
1.08 × 10-6
Cytidine
4.12 × 10-5
16
0.58
31.7
1.30 × 10-6
Thymidine
4.00 × 10-5
16
2.38
110
3.64 × 10-6
Table 2. Retention Times (t) of Nucleosides Derived from Unknown RNA Sample (min) and Quantitative Analysis Results for RNA Digest Mean Peak Height Concentrationa Peak # Mean t Identity (mm) (mmol/mL)
Figure 2. Chromatogram of an “Unknown RNA Hyrolysate.” C = cytidine (16.7%), U = uridine (22.5%), G = guanosine (22.2%), T = thymidine (5.4%), A = adenosine (33.2%).
Results and Discussion This lab exercise has been performed as part of a 4week/4-lab rotation. Each week 6 students perform the HPLC experiment, one student to a complete system. A larger number of students have performed the experiment in one lab period by grouping them on instrumentation: the data for standards are shared but each student is individually responsible for an unknown. The exercise can easily be finished in a 3-hour period. Standard samples are analyzed by each student in triplicate. The mean retention time for each individual component is calculated. For both separations all components elute in ≤ 6 min. From the known standard concentrations, mean peak heights, and detector attenuation, an instrumental response factor is calculated for each possible component. The unknown sample is analyzed in triplicate. The baseline separation for the components of each mixture is excellent. From the characteristic retention times determined for the standards and the corresponding peaks in the unknown, the components of the unknown are qualitatively resolved. From the mean peak height for each component of the unknown and the response factor for that component, the concentration of each compound in the unknown solution is determined. The mass of each component in the analgesic tablet is then determined. Analytical results for the percentage composition of each nucleoside residue in the unknown putative RNA sample are generally within 1.5% of the composition determined by mass during preparation of the “unknown” mixtures. A typical set of data for the RNA analysis is shown in Tables 1–3 along with a corresponding chromatogram of the sample (Fig. 2). For
50
1
0.55
cytidine
54.0
7.02 × 10-5
2
0.76
uridine
82.0
8.86 × 10-5
3
1.58
guanosine
67.6
8.86 × 10-5
4
2.26
thymidine
5.5
2.00 × 10-5
5
3.90
adenosine
43.4
1.40 × 10-4
aConcentration
= (Mean Peak Height) × (Response Factor)
Table 3. Comparison of Nucleoside Composition by HPLC vs Weight Nucleoside
% by HPLC
% by Weight
Cytidine
17.2
16.7
Uridine
21.7
22.5
Guanosine
21.7
22.2
Thymidine
4.9
5.4
Adenosine
34.4
33.2
their report, students are asked to discuss the implications of changing the mobile phase or the stationary phase. Given the nature of the C18 reversed-phase column, they are required to discuss the relative polarity of the nucleosides on the basis of their chromatogram. The results are quite convincing and meaningful to the students. Literature Cited 1. (a) Haddad, P.; Hutchings, S.; Tuffy, M. J. Chem. Educ. 1983, 60, 166–168; (b) Byrd, J. E. J. Chem. Educ. 1979, 56, 809. 2. (a) Parrett, D. In HPLC of Small Molecules: A Practical Approach; Lim, C. K., Ed.; Oxford, IRL: New York, 1986; pp 235–237; (b) Snyder, L. R.; Kirkland, J. J. Introduction to Modern Liquid Chromatography; Wiley: New York, 1979; pp 312–314; (c) Krstulovic, A. M.; Brown, P. R. Reversed-Phase High Performance Liquid Chromatography: Theory, Practice, and Biomedical Applications; Wiley: New York, 1982; pp 236–241. 3. A recent report describes the enzymatic digest of DNA leading to analysis of composition by HPLC: Wietstock, S. M. J. Chem. Educ. 1995, 72, 950. This report is complementary in that it requires no biochemical techniques and can be done simply in a 3-hour lab period.
Journal of Chemical Education • Vol. 74 No. 1 January 1997
