www.sge.

com

Improving Reproducibility
of HPLC methods

Q A
TECHNICAL ARTICLE
Solvents

In our lab we have many technicians and instruments. Despite documented methods we still find that different analysts still get different results. How can we improve the reproducibility of our HPLC analysis?
this reason the manner in which mobile phases are mixed is very important. Mobile phases can be prepared either by using a proportioning system to mix the solvents on a HPLC system or by preparing them individually. Modern HPLC pumps are able to mix solvents very effectively and reproducibly. Unfortunately, the composition of mobile phases mixed by different pumps can vary, and minor problems with the pump (such as a blocked filter) can cause variations in the mobile phase that are difficult to isolate. Therefore, premixing solvents is always recommended for optimum reproducibility. Even mobile phases prepared in the lab can be the source of irreproducible chromatography. Some laboratory workers are unaware that solvent volumes are not additive. For example, 600mL of acetonitrile topped up to 1 litre with water would have a different composition to 400mL of water topped up to 1 litre with acetonitrile. Generally a 60:40 acetonitrile:water mobile phase would be made up by adding 400ml of water to 600ml of acetonitrile, though this is not essential. What is important is that the actual method used to make up the mobile phase is detailed in the documented procedures.

Reproducibility of any analytical procedure is the primary concern of an analytical chemist working in quality control. To ensure good reproducibility laboratories document procedures for all of their routine analyses and yet inevitably experienced analysts get more consistent results than other workers. There is no substitute for experience, but detailed procedures can improve the overall performance of a laboratory.

Solvents come in many different grades and their purity varies between manufacturers and from batch to batch. While high grade solvents have less variation in quality even from different suppliers, the important point is to ensure consistency. To help achieve this it is useful to record solvent brand, grade, and lot numbers that are used with each HPLC instrument.

Mobile Phase Considerations

HPLC separations are very sensitive to variations in mobile phase composition. A 1% variation in a mobile phase composition will change the retention characteristics of a sample mixture. For

Figure 1. Comparison between two HPLC pumps mixing 60% acetonitrile with 40% water.

2
PUMP A

3 1 4

1. 2. 3. 4. 5.

Theophylline 4-Nitroaniline Methyl Benzoate Phenetole Xylene

1
PUMP B

2 3 4 5

2.5

5.0

7.5

10.0

12.5

15.0 min

Buffers
HPLC separations can be very sensitive to changes in pH and ionic strength. For this reason it is usually beneficial to buffer mobile phases to maintain ionic strength. This is especially important for the reversed phase analysis of polar molecules, as these can have irreproducible secondary interactions with many HPLC columns. Buffers are best prepared daily and should have at least 20mM concentration. Of course appropriate buffers should be chosen (see figure 2).

Temperature
Temperature variations over the course of a day have quite significant effects on HPLC separations. This can even occur in air conditioned rooms. For optimum reproducibility always use column ovens to overcome external temperature fluctuations. Column ovens are best operated between 35 and 40C.
Figure 2. Buffer Selection Buffer Phosphate pK1 pK2 pK3 Citrate pK1 pK2 pK3 Formate Acetate Tris Ammonia Borate Pyrrolidine Trifluoroacetic acid (TFA) Triethylamine (TEA) pKa 2.1 7.2 12.3 3.1 4.7 5.4 3.8 4.8 8.3 9.2 9.2 10.5 Buffer Range 1.1- 3.1 6.2- 8.2 11.3- 13.3 2.1- 4.1 3.7- 5.7 4.4- 6.4 2.8- 4.8 3.8- 5.8 7.3- 9.3 8.2- 10.2 8.2- 10.2 9.5- 11.5 UV Cutoff 190nm

Method Development
Gradient methods rely on pumps to mix solvents and are therefore less reproducible than isocratic methods. When gradient separations need to be used, make sure that they are developed for each instrument as there are always variations in how pumps deliver mobile phase gradients. When HPLC methods are developed for use by different workers on different equipment, it is important that the methods are robust. This means that the separation of key components should still be achievable if there are small changes in solvent composition, mobile phase pH, or in operating temperature. The method should also still work as the column ages. To ensure a HPLC method is robust, run standards with mobile phases that have compositions that differ by 2% from optimum to verify that satisfactory separations can be easily maintained. Of course it is important to verify that real samples can be effectively analyzed.

225nm

200nm 205nm 210nm 190nm 190nm 210nm 205nm 235nm

Columns
Use only columns that have been individually tested by the manufacturer as this ensures the column will be of a suitable quality. Batch tested columns can vary greatly in performance adding unnecessary complications to an analysis. Every SGE column is tested to ensure optimum performance. Make sure that the columns used are suitable for the analysis. Different brand columns with the same phase will give different separations. This doesnt mean that the same brand column must always be used for a given application. It just means that when columns are changed over it is important to verify that the column achieves satisfactory separation of key components. In many instances the separation can be improved by modifying the mobile phase slightly to match the characteristics of the column. Columns age with use, causing the separation to deteriorate over time. Peaks will broaden over time, due to loss of column efficiency. The retention times of peaks can decrease as the stationary phase gradually gets stripped off the support. These gradual changes are not noticeable over a series of samples, but may be noticed over a period of time. This is expected behaviour for any HPLC column and is not considered to be irreproducibilty as long as there are no variations over a set of samples.

T A D I AL

ED

Copyright 2001 SGE International Pty. Ltd. All rights reserved. TA-0104-H