Titration Rules – Sample preparation

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Whether you are titrating with a manual burette and indicator dye, or using a full scale automatic titration system, there are 10 main rules that successful scientists test by. These 10 rules fall into four categories: Sample Preparation, Analysis, Reviewing Results, and Maintenance and Upkeep. 

What Is Titration? Why Automate?

Titration is an analytical technique that is used for quantifying a number of chemicals across numerous industries. Manual titrations can be inaccurate and results can vary between operators based on the subjectivity of color indicators. Investing in an automatic titration system like the Hanna Instruments, HI932, is one step to greatly improve the accuracy and repeatability of titration results. However, the instrument itself is only part of the accuracy puzzle. Critical thinking and the application of best practices throughout the entire sample analysis process will further improve accuracy when using an automated system. This blog serves as a general guide to maximizing the accuracy of titration results by covering best practices for sample preparation, analysis, reviewing results, and maintenance and upkeep. 

Sample Preparation

#1 Choosing the Right Sample Size

Choosing the correct sample size is one of the most important criteria for ensuring that titration results will be accurate, efficient, and cost effective. Using a sample that is too small can yield inaccurate results because it offers very poor resolution in terms of data.  When data is limited, it is very difficult to interpolate where the true equivalent point is.  This results in poor repeatability and accuracy. Conversely, using too much sample will result in higher chemical costs due to excess titrant use, as well as excess chemical waste producted.The ideal sample size will consume titrant volume in the range 25-75% of the total burette volume.

You may be thinking – but how do we know how much sample to use to consume that amount of titrant? Luckily, if we know the estimated concentration of our sample, there is an easy way of determining the appropriate sample size based on titrant consumption using the titration equation! 

To quickly review, the titration equation is as follows:

CA = VT ∗ CT ∗ RR ∗ MM ∗ CF
CA is the concentration of the analyte (the unkown you’re trying to measure)
VT is the volume of titrant used to reach the end point
CT is the concentration of titrant
RR is the stochiometric reaction ratio of our analyte:titrant, in that order
MM is the molar mass of our analyte
CF is a conversion factor to adjust the results to your results units of choice (if necessary)
SA is the sample size of our analyte, and can be a mass or a volume

Luckily, we can rearrange the titration equation to solve for sample size. With a simple rearrangement, we can now easily solve for the appropriate sample size by plugging in our known values into the equation.

SA = VT ∗ CT ∗ RR ∗ MM ∗ CF

Let’s look at a real world example. Ketchup is a very popular condiment, and one component that is tested regularly is the salt content. To determine the appropriate sample size range for a titration of salt (sodium chloride—NaCl) content in ketchup (with an estimated 2% salt content), while using 0.1M (mol/L) silver nitrate as our titrant, and using a 25 mL burette. 

Applying the 25-75% rule to 25 mL (0.025L) burette, burette, we ideally would want to consume between 6 – 19 mL (0.006- 0.019L) of titrant.

Here is our equation, let’s define the variables and calculate both the upper and lower limits for sample size. 

SA = VT ∗ CT ∗ RR ∗ MM ∗ CF
SA is again our sample size, this is what we are solving for.
VT is our volume of titrant, for our lower limit, we have calculated that this is 0.006L
CT is the concentration of our titrant, so for this example 0.1 M (mol/L).
RR is our reaction ratio of our analyte, salt (NaCl), to our titrant, silver nitrate (AgNO3).  We get this information from the balanced chemical equation:
NaCl+ AgNO3 → AgCl +NaNO3
Since there are no coefficients before either our titrant or analyte, they are assumed to be 1. So, this equation is saying that 1 mole of salt will consume 1 mole of silver nitrate, making the ratio 1:1, which equals 1.
MM is the molar mass of our analyte, sodium chloride which has a molar mass of 58.44 g/mol (grams per mole).
CA is the concentration of our analyte which is estimated to be 2%, which is equivalent to 2g/100g

Now that we have defined the variables for the lower limits, let’s plug them into our equation.

SA = o.oo6L ∗ (0.1 mol ⁄ L) ∗ 1 ∗ (58.44 g ⁄ mol) ∗ 1(No conversion factor is needed.)
 (2g ⁄ 100g)

Canceling our units:

SA = o.oo6L ∗ (0.1 mol ⁄ L) ∗ 1 ∗ (58.44 g ⁄ mol) ∗ 1(No conversion factor is needed.)
 (2g ⁄ 100g)

Solving for SA:

SA = 1.75 g

Substituting our upper titrant limit of 0.019L as our VT in our equation, yields 5.555 grams.

Therefore, for this example, ~1.75- 5.55 grams of product is the ideal sample range which will consume enough titrant for good data resolution.

For additional assistance with determining ideal sample size – 
Contact a Hanna Instruments Expert Now!

#2 Using a Representative Sample

Sample size is not the only consideration for working with complex sample matrices. It is crucial to ensure that a representative sample be used for accurate determination of an analyte. A representative sample is one that embodies the sample matrix as a whole in that it contains all the parts all of the parent product or sample in the correct ratios. This is especially important for samples that are not homogeneous by default, such as spice blends or soil. If results are not repeatable, even when proper measurement technique is used, the likely source of error is a non-representative sample.

There be times when it is hard to guarantee a representative sample within the sample size range recommended for the method. If this is a case, a dilution is an excellent way to both assuring a representative sample, while still using a sample size that is suitable for good data resolution. A dilution is also a good idea if the sample size suggested by the previous section is too small to practically measure out. With a dilution, a larger amount of sample is weighed and added to a volumetric flask. Deionized water is added to the flask to bring the contents to the desired volume. The mixture is then allowed to stir for a period of time to become homogenized and or to extract the analyte. A small aliquot of this mixture is then titrated to an endpoint. With the Hanna Instruments automatic titration systems, the user is able to program the dilution into the titrator, so the results will be adjusted for the dilution factor. 

To program a dilution into the titrator you will need:

Size of Analyte to Be Diluted (Sample Size):  The mass or volume of the material added to the volumetric glassware
Final Volume:  The volume once the deionized water has been added to the sample
Aliquot size:  The volume of the sample that is being used for the titration.

#3 Using the Proper Measurement Tools and Techniques

Using the proper measurement tools and techniques are crucial components in the strategy to improve the accuracy of titration results. Recall from our titration equation, that the sample size is directly factored into the results. 

If the sample size that is input into the titrator is inaccurate, the titration result will be equally inaccurate. It is therefore important to ensure that you are able to obtain sample aliquots with the appropriate tools. Typically, liquid samples are measured out by volume and solid samples are measured by mass. 

Liquid Samples

Let’s begin by talking about liquid handling. Not all volumetric glassware was created equally, so it is important to understand the different types of glassware and their intended purposes.

Beakers and Erlenmeyer Flasks

Beakers and Erlenmeyer flasks, while they may contain graduated markings to indicate volume of sample, are primarily use for the holding, pouring, or mixing of solutions. They are typically not rated with an accuracy statement for measuring specific volumes. Using tools such as these to measure a sample size may cause fluctuation in results, thus hindering the repeatability of your testing. 

Transfer Pipettes

Disposable transfer pipettes (not to be confused with disposable volumetric pipettes) are another tool that may seem accurate due to their markings, but are generally not rated for accuracy. They are not recommended tools for volumetric measurement, but they are however, great tools for reagent addition that does not require precise addition.

Graduated Cylinders

Graduated cylinders are designed for measuring and pouring liquids. Graduated cylinders typically have an error tolerance of 1%, and are usually considered less accurate then volumetric flasks and pipettes. They are fast and easy to use, and can be a good choice for high throughput environments.    

Volumetric Flasks

Volumetric flasks are accurate for a specific volume of liquid. They do not typically have graduated markings to measure out different volumes of solution, but they are the glassware of choice for making accurate dilutions. 

Volumetric Pipettes

Volumetric pipettes typically offer the greatest amount of accuracy and are ideally suited for transferring liquids from once source to another. This type of pipette includes disposable plastic pipettes, glass pipettes, and auto pipettes. In order to achieve accuracy using these tools, it is crucial that the proper technique be used when sampling. The two most important factors are the aspiration angle and the immersion depth. When collecting a sample with a pipette, the pipette should be held vertically to ensure the proper amount of liquid is aspirated. The pipette should only be submerged in the sample enough to be able to aspirate the desired amount without pulling in air. Furthermore, the sample should be aspirated and dispensed a few times to prime the burette tip prior to transferring a final aliquot of sample to the titration vessel. When dispensing liquid from a pipette, the pipette should be held at an angle between 20-45 degrees directly over the center of the beaker. Care should be taken not to forcibly expel any remaining liquid from the pipette.

Liquid Handling Best Practices

Even among the same type of glassware, there is a class systems for quantifying accuracy.  Class-A glassware is the most accurate, and is usually accurate to two decimal places. This class of glassware will usually come with a certificate that specifies the accuracy of the tool.  Class-B glassware has higher tolerance of error than Class-A, and typically have an accuracy statement of one decimal place. As such, Class-A glassware tends to be more expensive than Class-B. For high accuracy needs, Class-A glassware is recommended and worth the investment. It is also helpful to use volumetric glassware that is geared toward the sample size that is being measured. Measuring 10 mL sample in a 10 mL graduated cylinder, will be more accurate then measuring 10 mL with a 100 mL graduated cylinder. 

In order to achieve accuracy when taking volumetric measurements, it is crucial to ensure that the volume is read correctly. Water tends to curve at the top of the volume, making it difficult to define the measurement. This curvature is called the meniscus. When reading the volume on volumetric glassware, the bottom of the meniscus should be on the marking of the desired volume. 

When adding a liquid sample to a beaker, make sure that the sample is added to the center of the beaker and that the sample is not stuck to the sides of the beaker. In most cases, a small amount of deionized water can be used to wash any remnants on the side of the container into the sample. 

Glassware should be rinse with deionized water and dried between samples, or if using an auto pipette, a new tip should be used for each different sample. All glassware should be cleaned with laboratory soap, acid rinsed (if necessary), and rinsed with deionized water before storage.

Some liquid samples are too viscous to be measure out accurately volumetrically. In these cases, mass can be used in lieu of volume. However, we have to be careful here because if our final units are related to volume, we have to account for the density of the sample in our results calculation for accurate computing. 

Solid Samples

Just like with liquid handling, it is important to use the proper tools and techniques when working with solid samples. 

Understanding the distinction between scales vs. balances is key. We often use the terms scale and balances interchangeably, but there are distinct differences between them. 

Scales tend to be able to handle a wide range of mass (both heavy and light). They lend themselves well to measuring ingredients or bulk product quickly. Scales are usually less expensive than balances. However, their open design and poor resolution do not make them suitable or ideal for measuring sample sizes for titration. They will introduce variability and thus affect your titration repeatability. 

Analytical balances are typically more sophisticated than scales. Additionally, they often have features such as shields to protect the sample from air currents that would otherwise cause the results to drift. Analytical balances also vary widely in terms of resolution and pricing so It is important to choose the correct analytical balance for your typical sample sizes. Below are the recommended resolutions based on the desired sample size.  

It is recommended that all balances are calibrated yearly.
Sample SizeBalance Resolution
1 gram0.1 grams
0.1 grams0.01 grams
0.01 grams0.001 grams
0.001 grams0.0001 grams

Solid Samples Best Practices

When setting up an analytical balance, choose a spot that is away from doors, fume hoods, and vents to further reduce the possibility of interference. In order to be accurate, balances should be leveled appropriately and calibrated per the manufacturer’s instructions. A set of weights can be purchased to ensure that the balance is reading properly. 

Balances should be tared, or zeroed, with the weighing vessel prior to the addition of sample.  For best results, take the mass of the sample directly in the titration beaker ensuring no product spills onto the balance.  If using a weigh boat, rinse the contents of the weigh boat 3 times with deionized water to ensure all product is accounted for. Since deionized water, in most cases, does not contain the analyte being tested, it can be added without fear of interference. 

You should be familiar with the accuracy of your measuring tools as the final calculated titration result will only be as precise as your least precise variable in the titration equation.

#4 Using the Right Type of Water

Just like with glassware, all water is not created equal. There are different classifications of water based on the purification process it undergoes. When preparing samples it is important to ensure that you have, and use, the correct water.

Tap Water:  

Tap water is the raw water that comes through the faucet from a private well or municipal source. Tap water contains all types of contaminants including minerals, disinfectants, and those which contribute to pH, acidity, and alkalinity. Due to the presence of potential contaminants, raw tap water is not recommended for lab analysis without further purification. Tap water, typically, has a total dissolved solids (TDS) level of 100-500 parts per million (ppm). 

Reverse Osmosis:

Reverse osmosis, abbreviated as RO water, is water that been purified through being pressure forced through a semipermeable membrane. Contaminants are trapped in the filter, whereas the clean water is allowed to pass through the membrane. RO water removes 98% of total dissolved solids (TDS), but does not remove all pesticides, solids, or VOCs.  RO water contains a TDS <100 ppm

Distilled Water: 

Distilled water, abbrieviated as DH2O, is water that has been purified through the process of distillation. Here, water is boiled, then the vapor is condensed into a sterile storage container, all while leaving solid contaminants behind. However, anything with a boiling point that is lower than water, like volatile organic compounds (VOCs), will be carried over into the distillate. Bottled water is not the same as distilled water, as it is often fortified with minerals. Distilled water has a typical TDS value of <0.5 ppm. 

Deionized Water:

Deionized water, abbreviated DI H2O, removes nearly all contaminants and is the gold standard of water for lab analysis. First, water is prefiltered through series of filters including physical, carbon, and reverse osmosis. The water then passes through cation and anion DI resins.  Here, positive and negative ions are captured and replaced with with H+ and OH ions, which combine to form pure water. Deionized water is typically measured using resistivity, and should have a value of at least 18 MΩ·cm.