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Is water grade really that important in Liquid Chromatography?

Part 1. Introduction

Water is one of the most important solvents commonly used in liquid chromatography applications. At present, across different laboratories various grades of water are used, either being commercially available and purchased or generated inhouse using commercial water purification systems. This practice is in a pursue of best possible solvent for a given analyses type. One must also consider the chemical properties of residual impurities within the water, including whether they are organic/inorganic, ionic/non-ionic, and UV absorbent or non-UV absorbent, when using UV detection. The application varies according to the factors listed above. Let us have a look at some points related to the grade of the water used in liquid chromatography.


Part 2. Water Used in Liquid Chromatography

Purified water specifically for HPLC use can be purchased rather easily today, as there is a variety of commercially available water from most of the solvents manufacturers. When you investigate the water bottle’s label, you will notice that it is intended for UV detectors, frequently used for HPLC analyses in many different applications/methods. To guarantee low absorbance water in the short-wavelength, the UV-absorbent organic matter is removed via distillation or some other methods. Therefore, it is always recommended to use the correct grade of water as the mobile phase when performing high-sensitivity analyses using a UV detector.

The water quality impact is particularly noticeable with gradient elution in reversed-phase separation modes. This is because the organic matter in the first liquid, which has a low elution capacity, is easily concentrated in the column, and as gradient elution proceeds, ghost peaks and increasing drift in the baseline appear. The Figure 1 below represents the baseline for the gradient going from 100% water to 100% acetonitrile in analyses where very low-quality water grade was used. As gradient elution progresses, many ghost peaks start to appear. Even with water purification systems, if the level of purification is low, or if the water purification system maintenance is insufficient and organic matter remains dissolved in the water, the same problem can occur. All this can results in higher TOC levels in water used for analyses.

Figure 1. Chromatographic baseline at 200nm – water 100% to acetonitrile 100% and back

Figure 1. Chromatographic baseline at 200nm – water 100% to acetonitrile 100% and back

Among the different water contaminants that may affect the quality of water used for sample dilution, standard solutions or mobile phase preparation, organics are probably the most important ones, as mentioned above. Figure 2 below represents chromatograms from an experiment where high purity (ultrapure) water with different TOC levels was used as mobile phase A with acetonitrile as mobile phase B; elution was a gradient employed on a C18 column and “0” volume injection principal was applied. It is obvious how increasing levels of TOC result in high chromatographic backgrounds. What is quite interesting is that it’s very unusual to find the TOC results on a bottled water certificate, nor it is typically mentioned on a specification sheets.

Figure 2. Chromatograms of ultrapure water containing different TOC levels.

Figure 2. Chromatograms of ultrapure water containing different TOC levels.

To further dispute the water impact on chromatography, I found very comprehensive study (source below) of the off-line TOC measurements of several brands of commercial HPLC-grade water. The study shows that TOC levels in purchased bottled water can reach levels as high as 700 ppb. Refer to table 1 below for the measured TOC values of bottled water compared with inhouse water generated using laboratory water system. In addition, to the table1, Figure 3 presents overlay of the chromatographic traces at 210nm for all 7x different types of water.

There is only one conclusion: contaminant peaks are present in most of the HPLC-grade bottled water tested. It is very clear that the high levels of TOC adversely affect the chromatographic baseline.

Water sourceTOC level (ppb)
Bottled water – A100.0
Bottled water – B87.0
Bottled water – C777.0
Bottled water – D16.5
Bottled water – E32.4
Bottled water – F25.5
Fresh ultrapure water (inhouse)7.0

Table 1. TOC (ppb) levels in various bottled HPLC-grade water­­ and inhouse generated water (source).


Figure 3. UV (210 nm) chromatograms of HPLC-grade water listed in Table 1.

Figure 3. UV (210 nm) chromatograms of HPLC-grade water listed in Table 1.

On the other hand, when performing measurements in the long-wavelength region of a UV (or VIS) detector, or when using another HPLC detector, such as a refractive index detector, spectrofluorometric detector, or conductivity detector, distilled water for HPLC is not necessarily the optimum choice. Of course, because it has a high level of purity, there are no problems in actual use. It may simply be unnecessarily expensive.


Part 3. Water for Ion Chromatography

In ion chromatography, any ion contamination in the mobile phase can cause noise and baseline fluctuation. Also, a “system peak” phenomenon can occur, with ghost peaks and negative peaks appearing in the baseline. This can potentially impact the accuracy of quantitative analyses. Therefore, care is required when selecting the right solvents, water including. In general, one should use ion-exchange water with a low residual ionic content with estimated conductivity not exceeding 1mS/cm (specific resistance no less than 1MΩ/cm). This type of water is considered appropriate for the mobile phase preparation.

It is known that carbonate ions are present at ppm level in commercial distilled water for HPLC and water that has been left exposed to air for some time. Another known fact is that also sodium ions are eluted if water is preserved in certain types of glass containers. All this must be carefully considered when selecting, handling and storing water for ion chromatography analyses.


Part 4. Water for Amino Acids

In the high-sensitivity analyses such as amino acids, performed using the post-column derivatization technique of amino groups with o-phthalaldehyde or ninhydrin, contamination of the solvents or mobile phase with amines or ammonia may cause ghost peaks and irregularity of the baseline (“ammonia plateau”). Ammonia may be dissolved in water that has been left exposed to air for some time, and there may be amines left in water with a low level of purification. Therefore, in amino-acid analyses, it is better to always use fresh water, if possible, that has been further purified using distillation.


Part 5. Summary

It is obvious that water grade and type have a vast impact on the chromatography, therefore prior next analyses have a think about what type of water you are going to use. With very high cost of laboratory investigations, it could be both effective and efficient to also investigate the quality of water used for the analyses; at least to maybe inject blank solvents alongside your sequence to have proof of your chromatographic trace.


Source:  https://www.merckmillipore.com/IE/en/water-purification/learning-centers/applications/environment-water-analyses/hplc/water-impact/mgWb.qB.gQ8AAAFAuXIQWTtA,nav?ReferrerURL=https%3A%2F%2Fwww.google.com%2F

Liquid Chromatography – Master the Basics

This article is part of our “Liquid Chromatography – Master the Basics” series, your go-to resource for comprehensive and insightful updates on the world of liquid chromatography. Each month in 2024 we will dive into a Liquid Chromatography topic, offering content that is both accessible to beginners and beneficial for experienced scientists.

Explore the Series

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Sebastian Jurek is an application consultant with Mason Technology with specialist knowledge in the Shimadzu range of instrumentation. He holds more than 22 years experience in chromatography techniques and analytical method development, optimisation and troubleshooting.

Get in touch with Sebastian today if you would like further information on our range of Shimadzu products.

Sebastian-Jurek-application-consultant-with-Mason-TechnologySebastian Jurek
Application Consultant for Shimadzu Chromatography
E:      sjurek@masontec.ie
M:     +353 87 436 4185
T:   +353 1 4154422

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