CHES Buffer Preparation & Usage Protocol | pH 8.6–10

CHES is a zwitterionic Good's buffer widely used in biochemical research to maintain stable pH in the alkaline range of 8.6–10.0.

Chemical Name

2-(Cyclohexylamino)ethanesulfonic acid

CAS Number

103-47-9

Useful pH Range

8.6 – 10.0

pKa (25 °C)

~9.3

Molecular Weight

207.29 g/mol

Ionic Interference

Does not chelate divalent metal ions; minimal UV absorbance at 260/280 nm

Materials & Reagents

Before beginning, gather the following materials:

• CHES (C110897) — Aladdin Scientific, ≥99% purity
• High-purity (18.2 MΩ·cm) deionized water
• 1 M NaOH solution (for pH adjustment)
• 1 M HCl solution (for pH correction if needed)
• Calibrated pH meter with electrode appropriate for alkaline range
• Analytical balance (±0.1 mg readability)
• Volumetric flask (100 mL or 1 L, as required)
• Magnetic stir plate and stir bar
• 0.22 µm membrane filter (if sterile filtration is required)

Step-by-Step Buffer Preparation Protocol

The following procedure describes preparation of 1 L of 100 mM CHES buffer at a target pH (e.g., pH 9.0):

1. Calculate and weigh CHES: For 100 mM, weigh 20.73 g of CHES (MW = 207.29 g/mol) on an analytical balance.
2. Dissolve: Transfer CHES powder to a 1 L beaker containing approximately 800 mL of deionized water. Stir until completely dissolved.
3. Adjust pH: Place a calibrated pH meter into the solution. Add 1 M NaOH dropwise with stirring to raise pH toward the target value (e.g., 9.0). Perform fine adjustment when within 0.2 pH units of the target.
4. Transfer and make to volume: Quantitatively transfer the solution to a 1 L volumetric flask and make up to the final volume with deionized water. Mix well.
5. Verify pH: Re-check the pH of the final solution; adjust if necessary. Note that pH is temperature-dependent — measure and record the temperature.
6. Filter (optional): For cell culture or sensitive assays, pass the buffer through a 0.22 µm membrane filter under sterile conditions.
7. Label and store: Label with concentration, pH, date, and operator name. Store at 4 °C for up to 1 month; discard if turbidity or precipitation is observed.

Note: CHES has a significant temperature coefficient for pKa (~−0.009 pH units/°C), so always adjust pH at the intended working temperature.

Troubleshooting & Critical Tips

• pH difficult to stabilize: Allow sufficient equilibration time after each NaOH addition (≥30 seconds). Ensure the pH electrode is freshly calibrated with buffers covering pH 7.0 and 10.0.
• Precipitation observed: CHES is generally highly soluble; precipitation may indicate contamination or use of hard water. Remake using 18.2 MΩ·cm water.
• Enzyme inhibition: CHES is compatible with most enzymes and does not chelate divalent cations (e.g., Mg²⁺, Zn²⁺), making it suitable for metalloenzyme studies.
• UV interference: CHES exhibits low UV absorbance near 260–280 nm, making it suitable for nucleic acid and protein quantification by absorbance methods.
• Autoclave compatibility: CHES buffer can be autoclaved; however, re-verify pH after autoclaving as slight shifts may occur.

Applications & Storage

CHES buffer is suitable for a broad range of research applications in the alkaline pH window:

• Gel electrophoresis: Running buffer for native PAGE and agarose gels requiring alkaline conditions.
• Enzyme kinetics: Reaction buffer for alkaline phosphatases, proteases, and other enzymes active above pH 8.5.
• Protein crystallography: Screening buffer in sparse-matrix crystallization kits for alkaline pH conditions.
• Western blotting: Transfer buffer component for alkaline transfer protocols.
• Capillary electrophoresis: Background electrolyte at pH 9.0–10.0.

Storage: Store dry CHES powder at room temperature (15–30 °C), protected from moisture and light. Prepared buffer solutions should be stored at 4 °C and used within one month. For long-term storage, aliquot and freeze at −20 °C.

Frequently asked questions

What is the useful pH range of CHES buffer?

CHES is effective between pH 8.6 and 10.0, with a pKa of approximately 9.3 at 25 °C. It provides excellent buffering capacity in the alkaline range not well served by phosphate or Tris buffers.

Does CHES interfere with metal ion-dependent enzyme assays?

No. Unlike EDTA-containing buffers, CHES does not chelate divalent metal ions such as Mg²⁺, Ca²⁺, or Zn²⁺. This makes it an excellent choice for metalloenzyme activity assays where metal cofactors must remain available.

How should CHES buffer be stored after preparation?

Prepared CHES buffer solutions should be stored at 4 °C and used within one month. For longer storage, aliquot the solution and freeze at −20 °C. Always inspect for turbidity or precipitation before use and discard if either is observed.

Why does my CHES buffer pH change after autoclaving?

CHES, like most Good's buffers, has a temperature-dependent pKa. During autoclaving, elevated temperature and subsequent cooling can cause a slight shift in pH. Always re-measure and re-adjust pH after autoclaving if precise pH is critical for your application.

Can CHES be used in UV-based spectrophotometric assays?

Yes. CHES exhibits minimal UV absorbance in the 260–280 nm range, making it compatible with absorbance-based nucleic acid and protein quantification methods such as NanoDrop measurements.

References

1. Good, N.E. et al. (1966) Hydrogen ion buffers for biological research. Biochemistry, 5(2), 467–477. doi:10.1021/bi00866a011
2. Sigma-Aldrich / Merck. CHES Product Information Sheet. (General physicochemical properties of CHES.)

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.