C HAPTER 7: S PECTROPHOTOMETERS & C ONCENTRATION A SSAYS Introduction to Biotechnology, BIOL1414 Austin Community College, Biotechnology Dept

L EARNING O UTCOMES Describe how a spectrophotometer operates, and give examples of their uses Explain the relationship between absorbance and transmittance in spectrophotometry Define the term pH and explain the relationship between the concentration of H + and OH - ions in acids and bases Describe the proper way to use pH paper and pH meters and which should be used in a specified situation Justify the need for buffers, describe how buffers are prepared, and calculate the amount of buffering agent needed when making a particular buffer Explain how protein indicator (solutions) are used Describe how VIS and UV/VIS spectrophotometers are used to measure protein or DNA concentration Note about this PowerPoint – There are several links in this PPT that allow you to explore more into different topics. Some of these links are animations, movies, or exercises. Please note, you must be in the slide show to activate the links. You can press F5 any time to active the slide show and “Esc” to exit.

U SING THE S PECTROPHOTOMETER TO D ETECT M OLECULES o Molecules are too tiny to be seen. o When an indicator solution changes color, that means a molecule of interest is present. A VIS spectrophotometer uses white light, composed of the visible spectrum, to detect colorful molecules.

Absorbance, Transmittance, and Reflection. A spectrophotometer measures how light interacts with atoms or molecules in a sample.

H OW THE S PECTROPHOTOMETER W ORKS Parts of a Spectrophotometer o Lamp o Prism o Sample holder o Display How a Spectrophotometer Works White light hits grating or prism Light is split into colors of the rainbow Wavelength knob directs different colors toward sample

How a UV Spectrophotometer Works. Similar to a VIS spectrophotometer, the UV spec shines ultraviolet light or visible light on a sample, and a detector measures the amount of light that passes through, or is absorbed by, the sample. Colors of Light in the Visible Spectrum. Humans can see light with wavelengths of about 350 to 700 nm.

How Concentration Affects Absorbance. If a sample has twice as many molecules as another, it can absorb twice as much light. This is true at any wavelength. It is important to know a sample’s wavelength of maximum light absorbance, so that the difference in absorbance due to concentration is obvious. λ max

I MPORTANT T ERMINOLOGY o Ultraviolet light – the high-energy light with wavelengths of about 100 to 350 nm; used to detect colorless molecules o Spectrophotometer – an instrument that measures the amount of light that passes through (is transmitted through) a sample o Nanometer – meters; the standard unit used for measuring light o Visible light spectrum – the range of wavelengths of light that humans can see, from approximately 350 to 700 nm; also called white light o Transmittance – the passing of light through a sample o Absorbance – the amount of light absorbed by a sample o Tungsten lamp – a lamp, used for VIS spectrophotometers, that produces white light (350 – 700 nm)

H OW TO U SE A S PECTROPHOTOMETER Explore! Click here! Explore! Click here! How to use a spectrophotometer How to use a spectrophotometer

U SING THE S PECTROPHOTOMETER TO M EASURE P ROTEIN C ONCENTRATION Proteins are too small to be seen, they must be visualized in a way other than direct observation. A spectrophotometer can assess changes in light absorbance due to protein molecule concentration and give numerical values to proteins in solution.

Determining the Wavelength of Maximum Light Absorbance. The lambda max for this sample is 440 nm. Once lambda max is determined for a molecule, the spec is set to that wavelength, and all readings for the molecule are made at that wavelength. The lambda max value is a characteristic of a protein.

U SING A S PECTROPHOTOMETER TO D ETERMINE P ROTEIN C ONCENTRATION : T HE B RADFORD A SSAY The Bradford Assay is a common assay used in biotechnology labs to determine the protein concentration of an unknown protein solution Prepare a series of dilutions of a known concentration of protein Mix dilution series and unknowns with the Bradford reagent Measure Absorbance at 595nm

S TANDARD C URVE Standard curve – a graph or curve generated from a series of samples of known concentration Plot a dilution series of known protein concentration and determine the linear regression Use the equation of the line to determine the concentration of the unknown solution.

T YPICAL B RADFORD S TANDARD C URVE

U SING A S PECTROPHOTOMETER TO D ETERMINE C ONCENTRATION OF N UCLEIC A CIDS If sample is pure (without significant amounts of contaminants such as proteins, or other nucleic acids), you can use spectrophotometer to measure amount of UV irradiation absorbed by the bases For quantitation of DNA or RNA, readings should be taken at wavelengths of 260 nm and 280 nm. The reading at 260 nm allows calculation of the concentration of nucleic acid in the sample, and 280 nm determines the protein contamination. 1 O.D. at 260 nm for double-stranded DNA = 50 ng/ul of dsDNA 1 O.D. at 260 nm for single-stranded DNA = ng/ul of ssDNA 1 O.D. at 260 nm for RNA molecules = 40 ng/ul of RNA Pure preparations of DNA and RNA have OD 260 /OD 280 values of 1.8 to 2.0, respectively.

N ANO DROP IT L IKE IT ’ S H OT The most common way to determine protein and nucleic acid concentration in the lab is using the NanoDrop spectrophotometer Advantages over the larger volume spectrophotometers are numerous Takes less volume More accurate Calculates the concentration for you Fast The NanoDrop vs. The Spectrophotometer The NanoDrop vs. The Spectrophotometer Watch this video on how to use the NanoDrop Watch this video on how to use the NanoDrop

P H & B UFFER S OLUTIONS

W ATER - T HE S OLVENT OF L IFE A solution is a liquid that is a homogeneous mixture of substances A solvent is the dissolving agent of a solution The solute is the substance that is dissolved An aqueous solution is one in which water is the solvent Most biochemical reactions occur in water

A CIDIC AND BASIC CONDITIONS AFFECT LIVING ORGANISMS o Water is in a state of equilibrium in which water molecules dissociate at the same rate at which they are being reformed o We can describe any solution according its H + concentration o Liquid water forms ions:

E FFECTS OF C HANGES IN P H Though statistically rare, the dissociation of water molecules has a great effect on organisms Changes in concentrations of H + and OH – can drastically affect the chemistry of a cell! Concentrations of H + and OH – are equal in pure water Adding certain solutes, called acids and bases, modifies the concentrations of H + and OH – Biologists use something called the pH scale to describe whether a solution is acidic or basic

A CIDS AND B ASES An acid is any substance that increases the H + concentration of a solution A base is any substance that reduces the H + concentration of a solution

T HE P H S CALE The pH of a solution is defined by the negative logarithm of H + concentration, written as pH = –log [H + ] For a neutral aqueous solution [H + ] is 10 –7 = –(–7) = 7

Bases donate OH – or accept H + in aqueous solutions Acids donate H + in aqueous solutions Acidic [H + ] > [OH – ] Neutral [H + ] = [OH – ] Basic [H + ] < [OH – ]

B UFFERS The internal pH of most living cells must remain close to pH 7 Buffers are substances that resist the change in pH minimize changes in concentrations of H + and OH – in a solution Most buffers consist of an acid-base pair that reversibly combines with H + pH can affect the function of a protein in solution Affects the ionization of the R-groups, this can alter 3-D protein folding, which can reduce function Therefore, when working with proteins it is important to work with buffered solutions

A N E XAMPLE OF A N ATURAL B UFFERING S YSTEM Buffer Systems in Human Blood: Carbonic Acid

M EASURING P H - E QUIPMENT Measuring the pH of a Solution pH paper pH meter Calibrating and Using a pH Meter It is important to calibrate the pH meter with appropriate buffer solutions. Typically, calibration buffer solutions are pH 4, pH 7 and pH 10. Calibrate a pH meter using at least 2 of these buffers

P H P APER pH paper is helpful when trying to quickly determine an estimate of pH This can be used by dipping the paper in a small aliquot of the solution and comparing the color strip to the color-code provided in the pH paper instructions

P H M ETER A pH meter is an accurate measuring device Can determine pH to 1/100 th decimal point (eg. pH=7.24) Uses calibration buffers Can be used to adjust the pH of a solution Can watch the pH of a solution change over time while adding in an acid or base pH meter does require upkeep and training. Periodically, the pH probe needs to be cleaned, solutions refilled, and/or replaced over time.

B UFFER P REPARATION 1. Determine the mass of each buffer component using the molar concentration equation 2. Add each mass of buffer solute to a beaker and add distilled water to approximately 75% of the final volume desired 3. Mix until dissolved (if possible, some solutes require pH adjustment to dissolve – EDTA) 4. Using a pH meter, measure the pH and adjust the pH up or down using NaOH or HCl 5. Transfer to a graduated cylinder and bring to volume 6. VERIFY the pH! Always, after bringing to volume

Y OUR T URN ! P RACTICE Q UESTION ! The formula weight of TRIS is g/mole. How is 100 mL of 0.02 M TRIS, pH 8.0, prepared?

B UFFERS IN A B IOTECHNOLOGY L AB Buffers are used in many applications in a biotech lab Many buffers have salt added to them Saline buffers include phosphate buffered saline (PBS), Tris-buffered saline (TBS) Frequently prepared as 10X stocks Most enzymes are sensitive to changes in pH Different buffer solutions are used for different applications – be sure to check the most appropriate buffer for your application Some buffers components can inhibit enzymatic activity Ion exchange chromatography buffers are very specific to the ion required in that chromatography procedure

Q UESTIONS AND C OMMENTS ?

R EVIEW Q UESTIONS Your Turn! Put your name at the top of a sheet of paper, answer these questions and hand in: 1. What is measured in a spectrophotometer? 2. What is the difference between a UV spectrophotometer and a VIS spectrophotometer? 3. What happens to the absorbance of a sample as the concentration of a sample increases? 4. If a sample has a pH of 7.8, is it considered an acid, a base or neutral? 5. If the pH of a hot tub is too high, say pH 8, then what should be added to bring it to a neutral pH? 6. Why must DNA and proteins be stored in buffered solutions? 7. How is Bradford reagent used to detect a specific protein in solution? 8. Which graph is used to determine the concentration of unknown protein samples?

R EFERENCES 1. Biotechnology: Science for the New Millennium Ellyn Daugherty.