흡광도에 영향을 미치는 요인 - heubgwangdo-e yeonghyang-eul michineun yoin

1. What are absorbance and transmittance?

흡광도(A)는 광학 밀도(OD)라고도 하며 요소가 흡수하는 빛의 양입니다.
빛이 물체에 부딪힐 때, 일반적으로 흡수된 빛의 파장이 물체의 전자 여기와 일치하기 때문에 분석 물질의 분자 또는 원자가 빛을 흡수할 수 있습니다.

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.

• 견본
  관심 있는 물질의 흡광도를 측정하기 위해 일반적으로 적절한 부피의 관심 분석물이 포함된 용액을 큐벳에 넣어야 합니다.
• 큐벳 재료
  세 번째로 중요한 것은 올바른 큐벳 재료를 선택하는 것입니다. 큐벳의 재료는 항상 투명하고 매끄 럽습니다. 연구원은 재료보다는 용액의 흡광도에 관심을 가지고 있기 때문에 최대 광 투과율과 적은 빛 산란을 보장합니다.

• 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.

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

• 큐벳 측면에 지문이 남으면 테스트 결과에 어떤 영향을 줍니까?
  큐벳 창의 지문은 대상 광선 투과를 방해하여 부정확한 측정을 유발할 수 있습니다. 지문이 빛을 흡수하기 때문에 약간 더 높은 흡광도 판독값이 나타나며 측정된 농도가 실제 농도보다 더 높아집니다.
• 광 경로의 교란이 있는 경우 흡광도 결과는 어떤 영향을 받습니까?
  빛의 경로에 어떤 것이 있으면 측정된 흡광도의 판독값이 증가합니다.
  

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.

• 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.

안토시아닌은 pH가 1.0일 때 2-페닐 벤조피란의 적색 형태이고, pH 4.5에서 무색 메탄올 형태가 염기성이며 전자 형태의 흡광도가 후자보다 훨씬 높다. 따라서 같은 용액이지만 pH가 다른 경우 흡광도 판독값이 다르기 때문에 농도가 달라지고 실제 농도가 같더라도 결론이 정확하지 않습니다.

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.

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

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

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.

큐벳 창에 있는 지문의 경우 적절한 큐벳 청소가 매우 중요합니다. 그렇지 않으면 실제 성능보다 더 높은 흡광도 판독값을 얻게 됩니다.
큐벳 창의 지문이나 기타 얼룩을 고품질 렌즈 용지 를 사용하여 닦아내는 것은 큐벳 창을 깨끗하게 유지하는 데 사용하는 것이 안전합니다.
주의사항 : 종이타월, Kimwipes 또는 이와 유사한 종이 제품을 사용하지 마십시오. 대부분의 종이 제품에는 연마제가 사용되기 때문에 큐벳의 표면이 긁힐 수 있습니다. 긁힌 자국은 빛의 산란을 일으키고 궁극적으로 측정에 영향을 미칩니다.

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.

따라서 큐벳을 위한 적절한 세척 솔루션은 사용 수명을 늘리고 보다 일관된 결과를 제공할 것입니다.