Welche Faktoren beeinflussen die Absorption?

1. What are absorbance and transmittance?

Die Absorption (A), auch als optische Dichte (OD) bekannt, ist die Lichtmenge, die von Elementen absorbiert wird.
Wenn Licht auf ein Objekt trifft, kann das Molekül oder Atom des Analyten das Licht absorbieren, normalerweise weil die Wellenlänge des absorbierten Lichts einer elektronischen Anregung im Objekt entspricht.

The rest of the light is transmitted, in other words, it passes through the object, which is called transmittance (T).
The more analyte is found in the solution, the lower transmittance will be due to the more light is absorbed by it.

2. Why measure absorbance?

In biochemistry, biology or chemistry, when analyte absorbs the light at a specific wavelength, a unique relationship exists between the individual atom/molecule and its UV Vis spectrum. This relationship can be used for:

• Qualitative analysis – determining the presence of certain substances.
  For example, determining pesticide residues in food, identification of contamination such as COD in water, identification of nucleic acid such as COVID-19 testing.

• Quantitative analysis – determining the amounts of certain substances.
  For example, determining the concentration of substances in air such as PM2.5, determining the concentration of harmful substance such as mercury and asbestos in makeup, measuring calcium and protein content in dairy products.

3. How is absorbance detected?

• Light source
  The all-important thing for light transmission or absorption measurement is the light source.
  Different light sources can be used for absorbance measurements. They differ in the spectral wavelength range, in their optical intensity and in the light stability.

• Probe
  Die Lösung mit den interessierenden Analyten im richtigen Volumen muss normalerweise in eine Küvette gegeben werden, um die Extinktion der interessierenden Substanz zu messen.
• Küvettenmaterial
  Der dritte wichtige Punkt ist die Wahl des richtigen Küvettenmaterials. Das Material der Küvette ist immer klar und glatt, um eine maximale Lichtdurchlässigkeit und weniger Streulicht zu gewährleisten, da das Interesse der Forscher an der Absorption der Lösung und nicht am Material liegt.

• The appropriate blank
  In order to correct undesired absorbance of absorbance measurements, such as light scattering, a blank is measured in parallel to samples. The appropriate blank includes all components except the analyte of the assay.
  Side Note: Particles in the solutions scatter the light, it will increase the measured absorbance value since they block the light path, as a result, less light reaches the detector.
• Detector of absorbance measurements
  A UV-VIS spectrophotometer is an instrument designed to measure absorbance in the UV-VIS region using the Beer-Lambert law. Measures the intensity of light passing through a sample solution in a cuvette and compares it to the intensity of the light before passing through the sample.

4.Theoretical background of absorbance measurements

• Detection path in cuvettes
  A solution contains interested analytes with known absorbance characteristics is placed into a cuvette. Then insert the cuvette into the instrument chamber, the absorbance reader will determine the light absorbance by calculating the optical density difference before and after passing the sample.
  (Light that does not pass through the detector is either absorbed or scattered. The scattered part can be corrected by measuring appropriate blanks, and is subtracted from this value to obtain real absorbance of the interested substance.)

• Absorbance in chemistry and life sciences
  After the absorbance measurement, the result is a value given in either transmission or optical density. Whereas, quantification of a substance in solution is the goal of the measurement, then the obvious question is how to convert the known signal into the concentration value. Generally, we can employ the Beer-Lambert law to get it.
• Beer-Lambert Law
  The Beer-Lambert law is very helpful as it allows quantification of absorbing substances without the need to add any other reagents, it describes the relation of absorbance, path length and concentration of an absorbing substance:
                                                                                   A=εlc
  A – absorbance
  ε – molar attenuation coefficient or absorptivity of the attenuating species – M‾¹cm‾¹
  l – optical path length – cm
  c – concentration of the attenuating species- M

It says that there is a linear relationship between the concentration, absorbance, path length and molar optical coefficient, which enables the concentration of solution to be calculated by measuring its absorbance.
For instance, in a standard cuvette the path length is 1 cm. For an absorbing substance and a specific wavelength, the extinction coefficient (ε) is a constant specific, usually the absorbance maximum of the substance, which provides information on how strongly the specific substance absorbs light at the specific wavelength.
As an example, the molar extinction coefficient for oligonucleotides is 32.4 µl*cm-1*µg-1. Therefore, a solution of 1 µg/µl oligonucleotides with a path length of 1 cm has an absorbance of 32.4 OD.

5.What will affect your absorbance measurements?

• Does cuvette material affect absorbance?
  When measuring in the UV-range, as it is required for nucleic acids or NADH, the UV-transparent cuvette is requisite to finish the measurement. Otherwise, you will get the maximum absorbance in all samples in this absorbance measurement because the material of cuvette absorbs UV light.

We have various cuvette material, and there are 5 common material you can choose to use, Plastic, Glass, UV Quartz (JGS-1), VIS Quartz (JGS-2), IR Quartz (JGS-3) cuvette, the same material has the same spectrum transmitting, but different fabrication has different light transmittance.
As an example, for our JGS-1 quartz glass material cuvette, Glued cuvette has 80% light transmittance, but the All Fused quartz cuvette has 83% light transmittance, and the highest applicable temperature is not the same.

• Does cuvette size affect absorbance?
  According to the Beer-lambert Law, when the extinction coefficient (ε) and the path length (l) are constant, the absorbance (A) is proportional to the concentration (c) of the sample. When the ε and the c are constant, the absorbance is directly proportional to the length of the light path, it is also equal to the inner width of the cuvette.
  The path length affects absorbance. With a longer optical path length, the light has to travel through more solution, and can hit more molecules or atoms, and be absorbed more.
  For a low concentration sample, the analytes may not absorb enough light with short path length cuvette, then the measurement is less effective.
• Will sample volume affect absorbance？
  For a standard cuvette, fill the cuvette about 80% full of the solution you wish to test is enough.
  But for a micro quartz cell or flow cuvette, and the sample you use is small enough to 10-400ul, then it’s important to make sure there is enough sample in the cuvette for the light to pass through. As we said before, the measurement will be less effective if the analyte can’t absorb enough light.
• Will Z-Dimension affect absorbance?
  8.5mm, 15mm, 20mm Z-Dimension are the common distance of cuvette center window to the cuvette bottom.
  If you use 15mm ZD black wall cuvette to a 8.5mm Z-height machine, the light beam will smission too low to tranmist the black wall of cell, which can’t reach the dector because it is blocked, then you will get 0% light transmission.
  
  We have varieties of absorption cuvettes with different ZD shown as below, such as standard pathlength, long pathlength, or flow cell.
  

  Z-Height	Type1	Code1	Pathlength1	Volume1	Type2	Code2	Pathlength2	Volume2
  8.5/15/20mm		QM79	10mm	50ul		QB41	10mm	100ul
  8.5mm		QB35	10mm	32ul		QF75	20mm	64ul
  15mm		QB44	10mm	50ul		QB43	10mm	200ul
  15mm		QM56	1/1.5/2/2.5/5/6.5mm	2/3/4/5/10/13ul		QB20	10mm	50/100/200ul

  
  There are more Self-masking cuvettes, please go to our web or contact us for quote if you want to buy cells.

• Wie würden die Testergebnisse beeinflusst, wenn Fingerabdrücke an den Seiten der Küvette hinterlassen würden?
  Fingerabdrücke auf dem Küvettenfenster stören die Transmission des Ziellichts und führen zu ungenauen Messungen. Dies führt zu einem etwas höheren Extinktionsmesswert und die gemessene Konzentration ist entsprechend höher als die tatsächliche Konzentration, da Fingerabdrücke das Licht absorbieren.
• Wie würden die Extinktionsergebnisse beeinflusst werden, wenn der Lichtweg gestört ist?
  Der Messwert der gemessenen Extinktion wird erhöht, wenn sich etwas im Lichtweg befindet.
  

Usually, these are air bubbles in the sample, condensation on a lid, dust, scratches or fingerprints on the window of the cuvette. Therefore, checking the cuvette just before measurement is recommended.
We have bubble-free New-Arrival cuvette to reduce the air bubble influence in the measurement.

Type	Item Code	Pathlength	Inner Width	Windows	Volume	Link
	QF61	10mm	10mm	4 Clear Windows	3.5ml	More Details
	QF62	10mm	10mm	2 Clear Windows	3.5ml	More Details
	QF47	10mm	10mm	4 Clear Windows	3.5ml	More Details

• How does pH affect light absorbance?
  When the pH of the solution changes, there are ionization in some of the molecules of the solution, then the structure of molecular changes, which will affect the determination of absorbance.
  As an example, for anthocyanin measurement of lycium ruthenicum, it is less accurate if measure the anthocyanin concentration in a single PH environment, because their color and absorbance changes with pH.

Das Anthocyan ist die rote Form von 2-Phenyl-Benzopyran, wenn der pH-Wert 1,0 beträgt, und die farblose Form von Methanol als Base bei einem pH-Wert von 4,5, und die Absorption der ersteren Form ist viel höher als die der letzteren. Daher ist bei gleichen Lösungen, aber unterschiedlichem pH-Wert die Konzentration unterschiedlich, da die Extinktionsablesung unterschiedlich ist, und die Schlussfolgerung wird ungenau sein, obwohl die tatsächliche Konzentration dieselbe ist.

As a result, pH-differential method is a better way to determine the concentration of anthocyanin of lycium ruthenicum.

• Does temperature affect UV Vis absorbance analysis?
  Temperature affects absorbance values.
  Different solvents’ interact performance are not exactly the same at different temperatures. It’s vital for an effective measurement to control the heating temperature especially some reaction undergoing at specific temperature. Solution parameters will change with the temperature changes:
  
  Rate of reaction-The reaction rate changes when temperature changed. This can cause a change in the activity of the sample. Enzymatic/biomolecular reactions are very sensitive to temperature.
  Solubility of a solute-Solubility is affected with variations in temperature. Poor solubility may result in imprecise absorption.
  Expansion or contraction of the solvent-This may lead to a change in the concentration of the solution and affect the absorbance, as absorbance is linearly related to concentration.
  Schlieren effect-This effect may occur with temperature changes, leading to a series of convective currents which may change the true absorbance.

For measurement of total nitrogen in water with spectrophotometer, in alkaline aqueous solution (>60℃), Potassium persulfate can be decomposed to produce potassium bisulfate and atomic oxygen, then the NO2-, NH4-, organic nitrogen in sample can be oxidized to nitrate nitrogen by atomic oxygen at 120-124℃. And we can determine the absorbance and concentration with this solution.

But there will be residual potassium persulfate in the solution to affect the absorbance if the temperature-controlled fail to meet the requirement. Potassium persulfate has a strong absorption peak at 220nm, which coincides with the absorption wavelength of total nitrogen. This absorption characteristic gradually weakens with the continuous decrease of potassium persulfate.

Temperature control can be achieved using high-performance nomothermal System for UV Vis spectrophotometry.

• How does stray light affect the optical density (OD) reading?
  Stray light is defined as light in the system at wavelengths (colors) other than the one intended, that is to say, stray light is not from the instrument’s light source and does not follow the optical path but reached the detector, which causing a deviation at the corresponding wavelength.
  
  Therefore, the optical density measured by the detector is higher than the true OD. In other words, the measured absorbance of samples is lower than the true absorbance due to the stray light contribution.
  There is one method to reduce stray light in these systems is the use of double monochromators.

6. How to reduce the effect on absorbance as mentioned above?

As we said before, different absorbance measurement requires different pH and temperature environment control, and stray light is a complex subject to figure out the exact problem to solve.

In this part, we will solve the influencing factors of cuvette for absorbance, right cuvette choosing, cuvette using and maintenance:

• How to choose the right absorption cuvette for absorbance studies?
  In our previous article, 7 Things to Think About When Ordering a Cuvette, we have attempted to provide answers to choose the right cuvette, it is specified in the aspects of cuvette material, pathlength, Z-dimension, volume, cover type, cell shape and etc.
  There will be a further explanation on path length choosing of cuvette in the following part:

• How to choose the right path length of cuvette?
  The proper pathlength of cuvette is to ensure absorbance values are within the dynamic range of the detector.
  Pathlength choosing of cuvette depends on the concentration of a sample and the chamber of instrument, which decide the upper light path length of detection.

• What time will use “long path” cuvettes?
  When the concentration of the sample is too low to be measured using a standard 10mm optical path length cell, and concentration is difficult in cases where the sample vaporizes or undergoes a chemical or molecular structural change during the concentration process, then a cell with a longer optical path length is used to enable the optimum absorption sensitivity of absorption measurement.

For instance, a well-known study using a long pathlength cell is the turbidity analysis of water. 50mm and 100mm cuvettes are often used for analyzing samples with a low turbidity.

There are cuvettes with optical path lengths of 20mm, 30mm, 40mm, 50mm, 100mm, and other long path length cells, made of JGS1 Quartz, 200-2500nm.

Path Length	Type1	Inner Width1	Code1	Link1	Type2	Inner Width2	Code2	Link2
20mm		10mm	QS43	Details		50mm	QC3909	Details
20mm			QF80	Details		20mm	QC3001	Details
30mm		10mm	QS30	Details		30mm	QR1103	Details
30mm		2mm	QB21	Details			QB26	Details
40mm		10mm	QS32	Details		10mm	QF13	Details
100mm		10mm	QS27	Details		φ19mm	QC2501	Details
50mm		20mm	QC51	Details		10mm	QC2801	Details
100mm		10mm	QC2701	Details		2mm	QC2401	Details

We also have long pathlength cuvettes made of optical glass, it range from 10-100mm. Click here to see more, and here to see the Glued* one .

• When will use “short path” cuvettes?
  With regards to some high-concentration samples, it allows measurement with a 10-mm standard path length cell after diluting.
  But for some high concentration samples and can’t be diluted easily, for example, due to the analyte may interact with the solvent, diluting a sample may cause a change in the absorbance (i.e., shift in the peak wavelengths). In this case, a short path length cuvette is used to measure high-concentration samples without diluting effectively.

For instance, A well-known study using a short path length cell is solution analysis in the NIR region. If a 10mm standard pathlength cell is used for absorbance measurement in the NIR region, saturation often happens because light absorption by the solvent, making it impossible to determine the absorption of the analyte. Then a short path length cell is used to prevent absorption saturation because of the solvent.

There are short-path cells with optical path lengths of 0.1mm, 0.2mm, 0.5mm, 1mm, 2mm, 5mm, and etc.

Path Length	Type1	Code1	Volume1	Link1	Type2	Code2	Volume2	Link2
0.1mm		QS41	35ul	Details		QF77	6ul	Details
0.2mm		QS23	70ul	Details		QF92	16ul	Details
0.5mm		QS31	170ul	Details		QM47	150ul	Details
1mm		QS21	350ul	Details		QC50	860ul	Details
2mm		QS26	700ul	Details		QF86	60ul	Details
5mm		QS24	1.75ml	Details		QC3701	650ul	Details

We also have short pathlength cuvettes with macro window, air-tight, or narrow width as below.

Path Length	Type1	Code1	Volume1	Link1	Type2	Code2	Volume2	Link2
0.2mm		QM27	60ul	Details		QF89		Details
1mm		QC47	350ul	Details		QM26	1.5ml	Details
1mm		QF11	350ul	Details		QM48	350ul	Details
2mm		QF12	700ul	Details		QM36	700ul	Details
5mm		QF14	1.75ml	Details		QC1401	1.75ml	Details
5mm		QM67	350ul	Details		QM8601	7ml	Details

We have more short path length cuvettes, such as 1.5mm, 2.5mm, 3mm, 6mm, and optical glass cuvette is also in stock. 

If you want to buy quality and affordable cuvette, please contact us or go to our web ecuvette.com.

After we studied the cuvette choosing of material, pathlength, volume, Z-Dimension and ordered the right cuvette, it’s time for us to learn how to clean the cuvette if we didn’t buy the disposable one.

• How to clean to the outer window of cuvette?
  The cuvette should be replaced immediately if there are cracks, chips or scratches on the window, or result in poor performance, less effective result and waste your sample and time, it is much expensive than the cost of a replaced cuvette.

Bei Fingerabdrücken auf Küvettenfenstern ist eine ordnungsgemäße Reinigung der Küvette sehr wichtig, da Sie sonst eine höhere Absorption als die tatsächliche Leistung erhalten.
Die Verwendung von hochwertigem Linsenpapier zum Abwischen von Fingerabdrücken oder anderen Flecken auf dem Küvettenfenster ist sicher, um das Küvettenfenster sauber zu halten.
Besonderer Hinweis : Verwenden Sie keine Papierhandtücher, Kimwipes oder ähnliche Papierprodukte. Denn bei den meisten Papierprodukten werden Schleifmittel verwendet, die die Oberflächen der Küvette zerkratzen. Kratzer verursachen Lichtstreuung und beeinflussen letztendlich die Messungen.

At the end of the day, clean your cuvettes thoroughly, air dry completely and store them in a cuvette rack or other suitable container. If the cuvettes didn’t dry completely and stored wet, they may dry with residue sample of previous application on the measuring surfaces, which will affect subsequent measurements.