pH – Samposium https://samnicholls.net The Exciting Adventures of Sam Mon, 27 Jun 2016 07:59:20 +0000 en-GB hourly 1 https://wordpress.org/?v=5.7.5 101350222 A pretend biologist’s guide to making an EDTA chelating (“buffer”) solution https://samnicholls.net/2016/06/26/edta-buffer-protocol/ https://samnicholls.net/2016/06/26/edta-buffer-protocol/#respond Sun, 26 Jun 2016 22:32:25 +0000 https://samnicholls.net/?p=1036 What the fuck is an EDTA chelating (“buffer”) solution?

EDTA buffer is a chelating agent that inhibits enzymatic degradation of DNA and RNA in a solution.
EDTA buffer is an agent that steals ions from molecular machines that need it to make DNA or RNA in a solution go bad.

What is it for?

Enzymes that modify, degrade and synthesize DNA and RNA usually require magnesium ions. EDTA buffer inhibits such metal-dependent enzymes by sequestering metal ions (primarily magnesium and calcium) from the solution. Thus EDTA buffer is a widely used component in buffers and solutions where there are biological products you wish to maintain the integrity of and/or reactions you may wish to suppress.

EDTA buffer is a component of TE Buffer (Tris/EDTA), a protective storage medium for DNA and RNA, and: TAE Buffer (Tris/Acetic Acid/EDTA) and TBE Buffer (Tris/Boric Acid/EDTA), both used for gel electrophoresis. EDTA buffer is also used as an anticoagulant for the storage of blood and preventing clumping of cells in liquid suspension.

Why do you say “buffer”?

Strictly speaking (as a pretend chemist for less than 24 hours), I thought a buffer was a solution designed to maintain a particular pH, whereas this buffer’s primary purpose is to sequester magnesium to suppress enzymes involved in degrading DNA. I find the term buffer a little confusing in this regard and feel chelator would be a better term. However, I’m horrendously unqualified and all the literature seems to refer to this solution as an “EDTA buffer”, but I just wanted to register my discontent1.

What do I need?

Reagents

  • EDTA disodium salt2 (FW 372.24)
  • Sodium hydroxide (FW 40) pellets
  • Milli-Q water (at least the volume of solution you wish to make)

Equipment

  • A suitable weighing scale (an analytical scale is preferred, but its maximum weight may be too low and you’ll require a top pan balance instead)
  • Realtime pH probe
  • Magnetic stirrer and flea

Glassware

The volume of your required glassware will depend on the volume of EDTA buffer you wish to produce. The values in brackets are the containers suggested for a recipe to produce 0.5M 200ml.

  • A volumetric flask capable of holding the exact target volume of your recipe (200ml)
  • A beaker capable of holding about 50-75% of the target volume of your recipe (250ml)
  • A bottle (Duran Flask) capable of holding the target volume of your recipe (250ml)
  • A bottle (Duran Flask) to store a suitable volume of MQ water (if not already available)

Bits and pieces3

  • Lab spatulas
  • Foil
  • Measuring cylinder (that can contain the volume of solution you wish to make)
  • Funnel
  • Access to an autoclave, and autoclave tape
  • Patience

What are those things?

EDTA

Ethylenediaminetetraacetic acid (widely referred to as EDTA because nobody can spell or say ethylenediaminetetraacetic) is a chelating agent, capable of sequestering metal ions (including calcium, iron and magnesium). The molecule is hexadentate (“six-toothed”): a cool word describing its claw-shaped structure that is capable of binding very strongly and very effectively to an atom in six places.

This feature of EDTA makes it rather ubiquitous and useful in many industries. For example, EDTA softens water to allow ingredients of soaps, shampoos and laundry detergents to work more efficiently. EDTA is also used to preserve and stabilise cosmetics, eye drops and skin care products in the presence of air, prevent discolouration of dyed fabrics in the production of textiles and can be used as a preservative for food (especially to prevent oxidative decolouration). EDTA can be used to treat many instances of heavy metal poisoning (lead, mercury and others) via chelation therapy, binding to heavy metals in the blood for safe excretion through urine. EDTA can also chelate excess iron from the blood, which can reduce the complications of blood transfusions. EDTA is used extensively in the analysis of blood, primarily as an anticoagulant.

EDTA is an essential medicine according to the World Health Organisation.

Sodium hydroxide

CAUSTIC
Sodium hydroxide is caustic to both metals and skin, and can cause serious eye damage.
In the event of exposure to skin, irrigate with water for 10-15 minutes.

Sodium hydroxide (familiarly named caustic soda) is a commonly used alkali with wide industrial applications including; pulping wood in the production of paper, refinement of bauxite ore to aluminium oxide for the production of aluminium, and the manufacture of soaps and detergents. Sodium hydroxide is also widely used in the preparation of foods, including chemical removal of skins from fruit and vegetables, processing of cocoa, poultry and soft drinks. According to Wikipedia, the unique crust of German pretzels and flavour of Chinese noodles are down to their preparation in sodium carbonate and lye-water respectively.

Sodium hydroxide is highly effective as an industrial cleaning agent, capable of dissolving grease, oils and fats. It is a common component of strong oven cleaner, glass and steel degreasers and drain openers – capable of hydrolysing hard to break down proteins in hair.

It is often used in the laboratory to as a means to raise the pH of solutions.

It is also used by serial killers to dissolve bodies4.

Milli-Q Water

Distilled, deionised and filtered water. Ion mass spectrometers will have trouble picking up more than a few parts per million. Really, just, really fucking pure water.

pH probe

An instrument for measuring the pH of a solution by measuring electric potential between two electrodes: the glass and reference electrode. Free ions in the solution (or lack thereof) cause a differential in charge across the inner and outer surfaces of a glass bulb which contains metal salts and surrounds the glass electrode. As the pH on the inside of the glass bulb (that surrounds the glass electrode) is known, pH can be quantified by measuring the differential in conductivity between the inner (glass) and outer (reference) surfaces of the glass membrane.

It is important to calibrate the pH meter before every use (or at at the start of the day, if it is to be used) to ensure accurate readings, as the calibration of the glass electrode will drift through use and time. Calibration should be done with at least two buffers (at either side of the scale of interest). Fancier models will also note the temperature during calibration to correct for variation in pH caused by temperature during actual use later. After use, ensure to follow instructions on cleaning the probe as the electrodes must be kept free of contamination. Typically after use a probe will be rinsed with deionised water, blotted dry and returned to its storage buffer (some form of neutral buffer that does not encourage ions to diffuse out of the electrode).

Solutions like EDTA buffer are only as good as the pH meter that they are buffered with, so it is important this instrument is well cared for. Ensure to follow product guidance for correct cleaning and storage of pH probes in both short and long-term, as these parameters vary between model and manufacturer.

Volumetric flask

A piece of glassware calibrated to contain a precise volume (at a particular temperature), used for precise dilutions and measures of stock solutions. I asked my supervisor why we have volumetric flasks when glass beakers and bottles are typically graduated with volume markings:

“Are they not that accurate then?”
“Awh jesus christ you may as well be measuring everything with your eyes closed!”

How do I make EDTA buffer happen?

Calculate recipe mass

The volume and concentration of EDTA buffer you want to make will depend on what you intend to use it for. For me, I was preparing an EDTA buffer as a component of TAE (Tris/Acetic Acid/EDTA) buffer for gel electrophoresis. A 50x TAE recipe requires 100ml of EDTA buffer at a concentration of 0.5M; a 10x TAE buffer will thus require 20ml. I settled for 200ml as a reasonable volume of EDTA buffer to make; not so much that it will sit in the lab for the next decade5, and not so little that the rather laborious effort will need to be repeated any time soon.

To determine the mass of EDTA required for the recipe, we work out how many moles of EDTA should be dissolved in our buffer to obtain the desired concentration (0.5M) in a given volume (200ml). You can adjust the equation below for your own recipe by altering the desired volume and concentration:

mol_{\text{EDTA in buffer}} = vol_{\text{buffer}} \times conc_{\text{buffer}}\\ \\ mol_{\text{EDTA in buffer}} = 0.2L \times 0.5M\\ \\ mol_{\text{EDTA in buffer}} = 0.2L \times \frac{0.5 mol}{1L}\\ \\ mol_{\text{EDTA in buffer}} = 0.1 mol

A 200ml solution of EDTA with a concentration of 0.5M will contain 0.1 moles. We can now derive the number of physical grams of EDTA that will yield this many moles using its molar mass6 as printed on the label7:

mass_{\text{EDTA for buffer}} = mol_{\text{EDTA in buffer}} \times \text{mol mass}_{\text{EDTA}}\\ \\ mass_{\text{EDTA for buffer}} = 0.1mol \times 372.24g.mol^{-1}\\ \\ mass_{\text{EDTA for buffer}} = 0.1mol \times \frac{372.24g}{1 mol}\\ \\ mass_{\text{EDTA for buffer}} = 37.224g

If you house a distrust for equations, we can also confirm this value with some empirical thinking given the molar mass of EDTA. Considering the molar mass of EDTA is equal to the number of grams required to make a 1 mole per litre (1M) solution, we can derive that the mass for half the concentration (0.5M) will be half the molar mass: 186.12g. For our recipe however, we do not wish to produce a litre, but 200ml. To maintain the desired 0.5 moles per litre concentration in a smaller volume (200ml) we require just a fifth (1L / 200ml = 5) of the already halved mass: independently verifying our value of 37.224g. Mathematics works! It’s important to note that 37.224g will produce a 0.1 moles per 200ml solution, which is another way of expressing 0.5 moles per litre, or 0.5M.

Pre-prep

  • Translate hieroglyphs on the Milli-Q water dispenser and dispense Milli-Q water into a Duran flask

Prepare the EDTA solution

Before you begin weighing, check that the sum of the mass of the beaker (or container) and the required mass of EDTA for your recipe is not likely to be greater than the maximum weight supported by the scale, because that totally didn’t happen to me.
  • Turn on your scale, wait until it is ready (it may beep, or just show zeroes)
  • Place your beaker on the scale, wait for the value to stabilise and tare the scale
Try not to disturb the surface that your scale is on. This includes writing in your lab book, or leaning against or touching the bench. These devices are incredibly sensitive! Analytical scales typically have glass doors to mitigate the effects of air currents (including your breathing), ensure these are closed when tare-ing the device, and reviewing the final weight.
  • Carefully spatula out the required amount of EDTA for your recipe and realise how futile attempting to measure out a mass correct to four decimal places is
Carefully is the operative word here. Any substance spilt or otherwise off-target from your beaker or weighing boat, will still be counted by your scale. To maintain accuracy you should clear up any loose substance once you’ve inevitably made a mess of this.
  • Move the beaker off, or out of your scales, and turn off the scale, transport the beaker to your magnetic stirrer
It’s good practice to cover the top of the beaker with foil if you are going to wander around the lab with it, or abandon it briefly while you work out how a magnetic stirrer works.
  • Add between half and three quarters of your target volume of Milli-Q water (e.g. ~150ml for our 200ml recipe) to the EDTA
The accuracy does not matter here, we’re just providing a solvent to dissolve the EDTA in. We’ll be topping up the solution accurately in a volumetric flask later.
  • Place your beaker on a magnetic stirrer, slip the “flea” into the solution, turn on the magnet and fetch your pH meter
Don’t turn the stirrer on too high, it will cause the flea to stop spinning smoothly and instead bounce around uncontrollably, potentially splashing your solution out of the beaker (or damaging the container).
Don’t panic when your EDTA does not begin to dissolve, EDTA is almost insoluble in water until the pH is increased to pH 8.0

Buffer the solution to pH 8.0

Doing this right takes fucking ages. Don’t start this at the end of the morning because you’ll miss all of the best sandwiches for lunch. Screwing this part up means starting over, so be patient.
  • Remove your pH meter from its storage buffer and calibrate it if necessary (see above)
Your buffers are only as good as their pH, look after your meter!
  • With your solution still stirring, insert the pH meter probe and wait for it to stabilise
  • Begin adding sodium hydroxide pellets one by one; each time, wait for the pH to plateau and stabilise before adding another
You’ll need approximately 18-20g of sodium hydroxide pellets per litre (so ~3.5-4g for 200ml)
Sodium hydroxide pellets are hygroscopic: absorbing moisture from their surroundings. Do not leave the container open to the air. Weigh out perhaps half as many pellets as you think you may need to prevent them all degrading too quickly. Do not worry about the pellets you are adding to the solution becoming wet and sticky, but do be aware that this small amount of moisture will compound across all pellets to increase the overall volume of the solution.
Sodium hydroxide is caustic, handle with care, especially once the pellets have absorbed moisture.
Do not just dump all your pellets into the solution, if you overshoot pH 8.0, you can’t just add an acid or more EDTA to bring the pH back the other way – you’ll have “used” some of the solution’s buffering capability, you’ll have to throw it down the waste sink. I totally didn’t do this either.
EDTA buffer is classed as non-hazardous waste and can disposed down the waste sink. Check the rules for your lab and don’t assume this is the case with other chemicals as it’ll probably get you fired, or kicked out of the lab.
As you add sodium hydroxide, the pH of the solution will increase, only to allow more EDTA to dissolve, reducing the pH again. Your solution is trapped in this knife-edge two-steps-forward-and-one-back pH push-pull game and is why the process takes so long.
You can also add sodium hydroxide solution with a pipette, particularly if you want more control over the process as you near the goal of pH 8.0. However, don’t forget that both sodium hydroxide solution and the water absorbed by the sodium hydroxide pellets will add to the overall volume of your solution. This volume must not exceed (and ideally should be comfortably below) the volume of buffer you intend to make in total.
  • As you add more sodium hydroxide pellets, the pH should slowly wave towards pH 8.0, at which point almost all of the EDTA should have dissolved
  • Once you have finally stabilised the solution as close to pH 8.0 as you dare, leave the stirrer and pH meter on for a few more minutes to verify your work
  • If you are sure and happy with the pH, turn off the pH meter, clean the probe (as per instructions) and return to its storage buffer solution
  • Turn off the stirrer, retrieve the flea, rinse, and return it to the flea storage box before it gets misplaced

Accurately raise to target volume

  • Pour the contents of the beaker into the volumetric flask
  • Rinse the beaker with a small volume of Milli-Q water, swirl and add this to the volumetric flask (repeat until the volumetric flask is filled to the graduation mark)

Sterilise

  • Transfer the contents of the volumetric flask to a suitably sized Duran flask
  • Give the Duran flask a suitable label (0.5M EDTA Buffer, your name, lab, today’s date, etc.)
  • Add a strip of autoclave indicator tape
  • Loosen the lid of the flask and sterilise the solution in the autoclave
Your vessel can shatter under the pressure of autoclaving if you leave the lid on tight. Once removed from the autoclave, do not seal until the solution is cool, as this may also cause your vessel to shatter.

What do I do now?

  • Tighten the lid only once the solution has cooled after autoclaving (to prevent the flask shattering)
  • Store at room temperature
  • Open and pour inside a laminar flow hood only, it’s a sterile product now!
  • Put a picture of the bottle on Twitter, well done, you are a god damn alchemist now

How do I fuck it up?

  • Waste time using a scale whose maximum weight limit is exceeded by your container and EDTA
  • Impatiently dump all your sodium hydroxide pellets into the EDTA+Milli-Q solution, ruin the buffer by overshooting pH 8.0 and have to start over
  • Use a poorly maintained or uncalibrated pH meter: the pH is probably the most important property of this product!
  • Blow up your hard work by leaving the lid on tight and exposing it to multiple atmospheres in the autoclave
  1. I suppose “EDTA chelator” makes it sound like it might chelate EDTA, which would only serve to cause more confusion. ↩
  2. EDTA seems to be most commonly distributed in a disodium salt form. Apparently it can be quite difficult to achieve full dissolution of EDTA in its disodium form and the tri- and tetra- salts are more readily dissolved. ↩
  3. These parts eventually become obvious, apparently. ↩
  4. See also, hydrofluoric acid occasionally used to dissolve bathtubs. ↩
  5. 200ml should provide enough EDTA chelator for 10L of 10x TAE buffer. Each litre of which will yield 10L of 1x TAE, which is in turn enough to fill a reasonably sized gel tank and make at least ten small gel slabs. This is a lot for just me, but in a communal lab, I’m sure we’ll find a use for my homemade TAE. ↩
  6. Which, rather helpfully, can be represented by the same fucking symbol as molar: M. ↩
  7. To confuse you more, it may be under Formula Weight, FW, Molecular Weight or MW. I am under the impression there are subtle differences between these terms that I haven’t quite got to the bottom of yet. ↩
]]> https://samnicholls.net/2016/06/26/edta-buffer-protocol/feed/ 0 1036