Calibration Data

A question that I seem to receive frequently: “Can you help me with the calibration?”

So, you have a sample.  And, there is some chemical in there (like glucose) and you want to know how much glucose is in the sample… maybe you have some idea, it should be less than something, right?  Were do you start?

Step 1: You select a test method.  You might choose HPLC, UV-VIS, FTIR, etc.
Step 2: Calibrate your machine.
Step 3: Measure your sample
Step 4: Ask, does the result make sense?

Calibrations are done to correct for machine errors and produce correlations that relate things (usually a signal value to a concentration).  Most lab equipment in common labs work with Light.  A UV-VIs (Spectrophotometer), Refractive Index detector, FTIR, Florescent detector, etc, all use light. The light passes through (or sometimes reflects off) your sample. The amount of the light that does or doesn’t make it past your sample is related to the concentration.

Most detectors have a “Linear Response Rage.”  This is the operating condition where the signal is directly related to the amount of material it is measuring.  And they have a “Lower Detection Limit” or the smallest amount that they can detect.  There is also the “Sensitivity” that is how well it can determine the small differences.

If your sample blocks all the light, the detector maxes out the signal.  No matter how much more you put in the way of the light, if all the light is blocked – all the light is blocked – your signal stays the same… at the max. You will need to dilute your sample and reduce the number of molecules that are blocking the light.

Now, if your sample is really dilute or the light is really bright that it doesn’t even notice the sample molecules then the detector says you have nothing there… This is the “Lower Detectable Limit.” The lowest concentration of molecules that are needed for the machine to notice. You might need to concentrate your sample, that is dry off the solvent to make is more concentrated.

Most machines are tuned towards the low end. That means they will detect a very low concentration of molecules. That setup sacrifices the detector’s upper limit. It will be saturated (all the light is blocked) at lower amounts of sample. You can fix this issue by diluting your sample to make it fit within the range that the machine can “see” well.  It is much easier to dilute a sample then try to dry it or concentrate it.

We call the typical operating range the “Linear Range.” If you plot the signal as a function of sample concentration, you will actually see a drop off in the signal on the far left, a zone that looks like a line, and a plateau where the light is blocked. See the picture below, if the signal is above 0.65 or below 0.1 the data can’t be used. Also, keep in mind that every machine, test dye, and procedure will require its own calibration curve… don’t use this one here for your data.

Operating ranges for a detector. You want to work in the linear range. That area is the most accurate.



So, how do you make the calibration standards?

Maybe you are measuring something easy, glucose… a standard HPLC + Refractive Index Detector (RID) and an H+ Column will work for this test.  Your samples are all in water.

Step A: Make a stock solution that you are absolutely certain of the concentration (that is grams per liter) and not amount (grams). These tests work on concentration not absolute amount. To make this solution, use a volumetric flask (these have a line etched in the glass to ensure the correct volume when full). Measure out an amount that your balance can detect (something like 1 gram) and put it into the flask. Now add water (at the temperature stated on the flask – the flask should state the volume of water at the line +/- the accuracy and the temperature for that volume). Now, add the water to the line. Cap. Mix. If you used a 100 mL flask and 1 gram of glucose you have a 10 g/L glucose standard. — By the way, bacteria and fungi eat sugar so, this standard won’t last more than a day or two!

Step B: Dilute the stock solution into known concentrations.  The simplest way is by factors of 2.  That is you take 10 mL of 10 g/L standard and add 10 mL of water.  Now you have a 5 g/L standard.  Take 10 mL of that into a new container and add another 10 mL of water. Now you also have a 2.5 g/L standard. Do this a few times… to create a set 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156  g/L  You need to use accurate volume measuring devices to do the dilutions. DO NOT WEIGH THE LIQUIDS (you added sugar to the stock, that changes the density and weighing it isn’t accurate unless you measure the density and apply a correction – Likewise make sure all the liquids are at the same temperature when you measure out the volume).

Step C: Test these samples and your unknowns (AT THE SAME TIME). If you are using a colormetric, or wet chemistry where your are measuring UV-VIS absorbance, record the absorbance number. For HPLC, you record the area under the peak (not the peak height). Since, test dyes, columns, light bulbs, your mood, can change with time, you want to test your standards at the same time you test your unknowns… no cheating…

Step D: Plot your data. You plot should look like the one above. If you use Excel, Gnumeric, OpenOffice, Apple Numbers, add a trend line to the linear range. The equation is called your calibration curve (even though it is linear – is called a curve). Normal convention puts the concentration as the x-variable (you set the concentration first) and the signal (or area) as the y-variable (that is dependent on the sample concentration). In practice, it is easier to have the signal (or area) as the x-variable and the concentration as the y-variable (this saves you from doing the algebra).

Step E: Use the calibration curve to convert the area or absorbance of your unknown to the concentration.  DONE!


I have included a Excel Calibration Workbook to help you.

Keep in mind that all these methods are based in concentration, not absolute amount. If you need to get to an absolute amount, you need to multiply by a volume term. So, you have a bottle of sugar that is 200 mL… (that is 0.2 L) and you used 10 uL to inject for the HPLC test.  The HPLC says it is 2.5 g/L — so, 2.5 g/L * 0.2 L = 0.5 grams sugar in the bottle + some amount of water.

Significant figures & Number of Digits: These are limited to the lowest accuracy device you used. So, if your volumetric flask was only good to 2 digits (+/- 0.02 mL at 20ºC ) your results are only good to 2 places 0.01 (you can’t go about adding more digits). If you think you need more accuracy you need to buy more accurate measuring devices… and then do multiple tests to find out how reproducible you or your machine is… +/- 0.001 is just about the limit for most normal things… if you see extra digits in someone’s data… question them as to why?

If you think you need help, message me on Research Gate.

Good Luck!



Travel – Day 3 – Drive

Today is Day 3 and the only plan for today is to drive.

The plan, go from Kansas City, Kansas to Alamosa, Colorado. That is 670 miles.  Take I-70 to Rt 40, then to 287, to 96 to Rt 10 and finally 160.

I-70 In Kansas

I-70 In Kansas.. bug splats included.

People, gas stations, lunch at McDonald’s, I-70 was like any other rolling highway.  Then we stopped, exited to get gas and turned onto Rt 40 West. No one… there were a few semi-trucks that were hulling grain. The route was paved, the roads off of it were not. It is understandable why you want a pickup truck here.

Keeping with the theme, we were being chased by a rain storm.  The difference is that you can see it.  Way off in the distance, a few miles away, gray clouds and dark streaks reach down and touch the ground. Drive faster and avoid the rain.  We were basically alone on the road for about 5 hours.

Kansas Rt 40

Kansas Rt 40

There really isn’t a single good photo to describe driving through the miles of land used to grow food. The entire road was setup for some type of grain or corn or something, every so often we would pass by a silo and maybe a truck was there to hull it out.  There were no tourists, no gas stations, spotty cell service at best, no tow trucks, or rest stops.  Every hour or so, you would find what looked like a shop or town… A single building, maybe school, maybe a gas station with a sign on the door to call for someone to come by and assist you.  This a clear reminder of how much land is needed to feed a country, or two.  Imagine living at work, miles away from the nearest shop, growing crops.

Eventually, we get closer to the mountains of Colorado. At first the border between the states is not even noticeable.  Then, you start to see the mountains off in the distance. The land used for crops becomes more scarce and the ranches start to take form. We drove along an old railroad, down back road and through places the genuinely looked abandoned.  Periodically, there would be a house, a trailer, or a truck parked a few miles away from the road. I presume they are living there.

Walsenburg, Colorado

Photo from Walsenburg, Colorado

Driving through the mountain roads was a challenge for the car.  Before we left, Stephen had the oil changed, new air filter installed, new transmission filter and fluid, coolant flushed, and we changed the spark plugs. Going up the hills… there was no power. The car was struggling at the high altitude.  We reached Alamosa at about 7:30 pm and checked into the hotel. Dinner at Chilli’s.  Since the car was having issues, we decided to change the plan and take the car to the dealer and check to see if something was not working with the transmission or spark plugs.

Tomorrow’s plan. Get car checked at 7:30 am…

Travel – Day 2 – St. Louis

Today we departed from the Ohio/Indiana border and drove West. The goal for today?  To tour the “Gateway to the West.” That is the St. Louis Archway. This is a 630 ft tall stainless steal monument that was built to signify the westward expansion. We drove about 6 hours, non-stop through Indiana and Illinois.

Day 2 - Indiana

Day 2 – Indiana

In Indiana, Illinois and Missouri all lanes move at the same speed and drivers will occupy any free lane at what ever speed they please… Very interesting.

Motor cycle drivers in left lane.

Motor cycle drivers in left lane.

Rain was a common theme for our trip.

Rain was a common theme for our trip.


Illinois - Typical Drive

Illinois – Typical Drive


We reached the Arch and took the ride to the top. This 5-10 minute ride took a few hours of preparation. The cars are spaced apart as to limit the number of people reaching the top of the arch and allow for viewing time. The entire area is under construction and the water front spaces (such as the parking deck we put our car in) were flooded and under water. In a few years there will be a new visitor center and museum. In the mean time, ticket sales are in the old courthouse building, about a 10 minute walk from the arch. Keep in mind the road closings. As we learned, with all the construction, there are lots of walkways and roads that you can not pass thorough. It took a few tries to find our way about the city.

St. Louis Arch and Construction Site

St. Louis Arch and Construction Site

At the top of the arch are a few viewing windows flanked by people. You know, when you are at the bottom, you look up and you can almost see the arch moving about… well, at the top, you can feel it moving…

As we were leaving the park, where the arch is located, one of the horse and buggy drivers yelled out to us. She informed us that a big storm was on its way, we had about 10 minutes, they were being called in and she noticed that we didn’t have an umbrella… Very nice person… It turned out this storm would produce funnel clouds and tornado warnings.  We managed to leave and head to a Cheesecake Factory for dinner in Chesterfield. By the end of dinner, the sirens were blaring, the storm was raging behind us.  We drove West.  Just barely on the edge of the cloud front… Had we stayed in St. Louis for dinner, we would have been trapped in the storm.

Dark Storm Clouds in St. Louis, looking east from Chesterfield. Tornado sirens in the background.


After running from the storm, our check point was Kansas City.  We would drive for about 250 miles before putting in for the night.

Travel – Day 1 – Ohio

This summer, we are traveling through the Western part of  the U.S. Starting from Ohio, we set out west to visit family and friends at the very edge of Ohio. This is area that is close to New Bremen and Shelby County. Since we live close to Pennsylvania, this was a long drive through route 30… With lots of rain…


Did I mention that we had lots of rain… The fields were soaked.


One key highlight was the Maria Stein Countryfest in Maria Stein, Ohio. This community wide picnic featured Tractor Square Dancing. Where a group preforms a square dance on tractors. That is, driving tractors to mimic square dancing. The thrill is watching the skill level of folks ride tractors at high speeds in circles. This years, it was wet. It had been raining and the mud was scraped off into piles around the dancing “floor.”



Polaroid – Back in the 1960s…

Imagine you are living in the 1960s and you want to take a photo.  As you know, you are going to take a few photos, put the film in the mail or drop it off at your local photo lab and wait. Suppose, you don’t want to wait for the film to be sent out and developed… So, you pull out your new Polaroid Land Camera with Instant Picture Development.


This is one of my more favorite cameras to operate. The camera has a film pack inside. This film pack also includes the developing solution in little pockets. When you pull out the film and print sandwich the chemicals develop the print. Polaroid stopped making film and went under a few years ago. However, FujiFilm still makes the FP-100c color film packet for this camera, which you can buy on Amazon. Each photo is about 90¢. To take a photo, there are 4-ish steps…


Open the camera and find something to take a photo of…

Polaroid Land Camera Open

When you look through the viewfinder, you will see these little arrows and diamonds to tell you where the edges of the picture will be. Move the two levers to set the arrow to the approximate distance of the item. The levers are right where your index fingers will be, good product design!



Polaroid Land Camer Viewfinder Image

Adjust your scene.  Click the shutter button. The internal battery and electronic shutter will attempt to correct for sunlight. If not, there is a manual aperture adjustment on the lens.

Now, the fun part. There is this little white tab on the right of the camera, in front of a small trap door. Pull the tab. Slow, smooth, with force. Don’t Yank On It.

Polaroid Land Camera Pull Tab

Polaroid Land Camera Film Tab

This smaller tab will cause the door to open and another larger tab will come out from the door. In a single, smooth motion, pull out the larger tab. The big tab is the one that applies the chemicals to your photo and moves out the print. It is attached to the paper that will drag out the negative and the print.


Polaroid Land Camera - Film Out

Now, you wait.  There is instructions printed on the tab (see the words in red) that tell you wait a few minutes for the print to develop. You will adjust the time to wait based on the temperature… The biggest mistake is not waiting long enough… Then peel off the print from the paper and chemicals.  Discard the chemical covered negative.









Don’t touch the print for a few minutes. It needs to dry.

DONE! Instant photo.

The thing that I like the most is the color. These photos tend to be very saturated. Deep blacks with minimal detail in the shadow and vibrant colors that almost pop off the page.  They always have a clean look that captures the colors almost perfectly (something that digital has difficulty doing). The photos are shiny… high gloss.

Polaroid FujiFILM

FujiFILM print from a Polaroid Land Camera. Scanned with Epson V500 (dust included).




Gas Mileage Tracking

Question: Commute to work 100 miles/day (total), how much do you spend on gas?

Answer: For a 2004 Chevy Impala with 28 ± 2 MPG, it is $0.08 ± 0.01 per highway mile.


This table shows the amount of gas, the purchase location, MPG and cost per mile for operating a 2004 Chevy Impala... most of these values are from highway driving.


By Vendor

Company Avg MPG
Sheetz 28.35
BP 28.58
Petro 27.16
Pilot 28.82