Water Used for HPLC Analysis

High-performance liquid chromatography (HPLC) is a widely used analytical technique that is used to separate, identify, and quantify chemical components in a wide range of samples. HPLC relies on the use of a liquid mobile phase and a stationary phase, typically a column packed with a stationary phase material. The choice of the mobile phase is critical to the success of an HPLC analysis, and water is often used as a key component of the mobile phase.

Water is used for HPLC for several reasons. First and foremost, water is an inexpensive and readily available solvent that is compatible with many analytical samples. Water is also a polar solvent, meaning it can dissolve a wide range of polar and ionic compounds. This makes it an ideal solvent for many HPLC separations, as many target compounds are polar or ionic in nature.

However, the use of water for HPLC is not without its challenges. Water is a highly polar solvent, and as such, it can interact strongly with the stationary phase of an HPLC column. This can lead to peak broadening, poor resolution, and decreased sensitivity in HPLC separations. To combat this, various additives can be added to the mobile phase to modify the properties of the water and improve chromatographic performance.

One common additive used in HPLC mobile phases is an organic solvent. Organic solvents such as methanol, acetonitrile, and ethanol can be added to the water to reduce its polarity and improve the separation of non-polar compounds. These solvents also have the added benefit of reducing the interaction between the water and the stationary phase, which can lead to improved peak shape and resolution.

Another common additive used in HPLC mobile phases are buffers. Buffers are used to control the pH of the mobile phase and can improve the separation of acidic or basic compounds. Buffers can also help to prevent changes in pH that can occur during the analysis, which can lead to changes in the retention time and peak shape of target compounds.

In addition to these additives, water used for HPLC must also meet certain purity requirements. Impurities in water, such as ions, dissolved gases, and organic contaminants, can negatively impact the performance of an HPLC analysis. To ensure the purity of the water used for HPLC, it is often purified using various techniques, such as reverse osmosis, deionization, and distillation.

In conclusion, water is a commonly used solvent in HPLC, thanks to its availability, compatibility with many samples, and polarity. However, the use of water in HPLC can lead to challenges such as peak broadening and poor resolution. To combat these challenges, additives such as organic solvents and buffers can be used to modify the properties of the water and improve chromatographic performance. Additionally, water used for HPLC must meet certain purity requirements to ensure optimal performance.

Types of Water used in HPLC analysis
When it comes to High-Performance Liquid Chromatography (HPLC) analysis, water is a commonly used solvent in the mobile phase. However, not all water is created equal, and the quality of the water used in HPLC can have a significant impact on the accuracy and precision of the analysis. In this article, we will discuss the different types of water used in HPLC analysis.

1. Tap Water
  • Tap water is the most commonly available source of water. However, it contains impurities such as chlorine, heavy metals, and organic compounds, which can affect the HPLC analysis. Therefore, tap water is not suitable for HPLC analysis without proper purification.

2. Deionized Water
  • Deionized water is water that has been purified by removing all ions, including minerals and salts. This type of water is suitable for HPLC analysis as it is free from ionic impurities that can interfere with the separation of target compounds.

3. Distilled Water
  • Distilled water is produced by boiling water and collecting the steam, which is then condensed back into liquid form. This process removes impurities such as minerals and salts, as well as microorganisms and organic compounds. Distilled water is suitable for HPLC analysis as it is free from impurities that can interfere with the separation of target compounds.

4. Reverse Osmosis (RO) Water
  • Reverse osmosis is a water purification process that uses a semi-permeable membrane to remove ions, molecules, and larger particles from water. RO water is free from impurities such as minerals, salts, and microorganisms, making it suitable for HPLC analysis.

5. Ultra-Pure Water
Ultra-pure water is water that has been purified to a higher degree than deionized or distilled water. It is produced using a combination of techniques such as reverse osmosis, ion exchange, and distillation. Ultra-pure water is free from all impurities, including microorganisms, particles, and organic compounds. This type of water is typically used in sensitive analytical techniques such as HPLC, where the purity of the solvent is critical to the accuracy and precision of the analysis.

What level of purity is required for HPLC analysis?
The purity of the solvent employed in High-Performance Liquid Chromatography (HPLC) analysis is crucial to the analysis's accuracy and precision. In general, the solvent's purity should be at least 99.9%. In some circumstances, though, higher purity levels may be required.

Impurities such as ions, bacteria, particles, and organic molecules can reduce the purity of the solvent used in HPLC analysis. These contaminants can interfere with target compound separation, resulting in erroneous and imprecise findings. As a result, it is critical to employ an impurity-free solvent to assure the accuracy of the analysis.

The amount of purity required for HPLC analysis is determined by the detection method's sensitivity and the type of the substances being tested. A high-purity solvent, for example, is necessary for the analysis of trace-level contaminants in pharmaceuticals to ensure precise and consistent results. Typically, ultra-pure water or high-performance liquid chromatography grade solvents are employed in this scenario.

The purity of any reagents employed in the analysis, such as buffers, should be evaluated in addition to the purity of the solvent. Impurities that can interfere with the separation of target chemicals, such as metal ions and organic compounds, should be avoided in HPLC buffers.

Finally, the solvent purity level employed in HPLC analysis should be at least 99.9% to ensure precise and dependable results. However, depending on the sensitivity of the detection method and the type of the molecules being tested, the level of purity required may vary. To ensure the integrity of the analysis, high-quality solvents and reagents must be used.

What Kind of Water Should Use for Reversed Phase Chromatography?
Since reversed phase chromatography is typically used as a technique for analyzing or separating organic substances, the water used to prepare mobile phases or samples should be sufficiently free from any organic matter, but the purity level required depends on the specific detector used.

If using an ultraviolet absorption detector, it is normally recommended to use commercial HPLC grade distilled water. Impurities with absorbance in the UV region are removed from commercial HPLC grade distilled water and absorbance levels in the shorter wavelength regions are guaranteed. Therefore, HPLC grade water generally can be used without worry. If you purify water yourself, however, distillation and ion-exchange filtration alone are normally insufficient. 

Instead, an ultra-pure water production system is strongly recommended. In recent years, the trend has been to use ultra-pure water production systems that can reduce the total organic carbon content (total organic carbon or TOC) by using ultraviolet irradiation to decompose organic matter. In particular, in the case of gradient elution, the difference in TOC levels can significantly affect the appearance of ghost peaks. 

On the other hand, if a fluorescence detector and mass spectrometer are used, commercial HPLC-grade distilled water may be inadequate. In such cases, either use fluorescence analysis grade or LCMS grade water or use an ultra-pure water production system. 
  1. Purified water with an extremely low TOC value and resistance of 18 MΩ·cm or greater (electrical conductivity of 0.056 μS/cm or less).
  2. Water purified using a combination of methods, such as reverse osmosis membranes, ion-exchange polymers (including continuous ion-exchange substances), activated carbon, ultraviolet rays, and ultrafiltration, so that resistance is 18 MΩ·cm or greater (electrical conductivity of 0.056 μS/cm or less).

What Kind of Water Should You Use for Ion Chromatography?
Given that ion chromatography is essentially an analytical technique for detecting ionic compounds, the most crucial point to remember is to utilize water that has had all ions removed. Water from an ultra-pure water production system with a specific resistance of 18 Mcm or more is preferred.

As previously stated, the purity of commercial HPLC grade distilled water is guaranteed for use in analysis utilizing UV absorption detectors, however it is not guaranteed for use in assessing inorganic ions. As a result, before utilizing the water, ensure that it does not cause any analysis issues.

In general, commercial water marked as ion-exchanged water or purified water is not recommended, because the quality can vary depending on the manufacturer. Based on the above points, it can be said that for ion chromatography, using freshly purified water from an ultra-pure water production system will provide more trouble-free and worry-free results.

What Is the Correct Way to Use Water?
The better the purity of water, the more care must be taken when handling it. Because water may dissolve a wide range of compounds, contamination begins the moment it comes into contact with the air. If you use commercial water, drink it as quickly as feasible. It is critical to maintain ultra-pure water production systems on a regular basis. When purchasing commercial or refined water from an ultra-pure water production system, it is also critical to minimize contact with air by not producing bubbles. Additionally, try to avoid using wash bottles and instead add water directly into the preparation container.

Water in wash bottles can become contaminated easily and may contain plasticizers leached from the plastic bottle inner walls. (Replace water in wash bottles often.) Needless to say, always pre-wash equipment with fresh water before use.

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