Chlorophyll a/b Ratio as a Criterion for the Reliability of Absorbance Values Measured for the Determination of Chlorophyll Concentration

엽록소 농도 결정을 위하여 측정한 흡광도 값의 신뢰도 검정 지표로서 엽록소 a/b 비례치

Abstract

The Beer-Lambert law states that absorbance is proportional to the concentration of a solute in a solution at a given wavelength. This linearity works for an ideal or a 'sufficiently diluted' solution, so this linearity is often used as a criterion for the fidelity of the absorbance value measured. In this study, we used a chlorophyll (Chl) solution, isolated from rice leaves with 80% acetone to test the use of the Chl a/b ratio as an additional criterion for checking the fidelity of measured values using four different absorption spectrophotometers: Cary4E, UV-1650PC, Versamax (a microplate reader), and NanoDrop 1,000(which can handle a $4{\mu}l$ aliquot). We used Chl solutions of varying concentrations from $0.2{\mu}g/ml$ to $200{\mu}g/ml$ to measure absorbance values at 645 nm and 663 nm and checked the linearity first. The results indicated that the range of Chl concentrations that we can rely on based on the linearity was similar to the range in which the calculated Chl concentrations based on the measured absorbance values agreed with the known concentrations. However, some border cases or cases with very low Chl concentrations inside the fidelity range of Chl concentrations did not agree with the criterion that the Chl a/b ratio should not change after dilution of the Chl in the solution. These results suggest that the Chl a/b ratio is a better criterion for the reliability of the absorbance values measured for the determination of chlorophyll concentration than the criterion based on the linearity suggested by the Beer-Lambert law.

비어 람버트 법칙에 의하면 충분히 묽은 용액에서 주어진 파장에서 측정한 흡광도는 용질의 농도에 비례한다. 반대로 충분히 묽은 용액이란 측정한 흡광도 값이 용질의 농도에 비례관계를 가지는 범위에 있는 용액을 말하지만 실제 확신하기가 어렵다. 본 연구에서는 벼 잎에서 추출한 엽록소 80% 아세톤 용액을 사용하여, 측정한 흡광도 값의 신뢰도를 판단할 수 있는 기준으로서 엽록소 a/b 비례치의 사용 가능성을 4가지 다른 흡광분광분석기(Cary4E, UV-1650PC, Versamax, NanoDrop 100)을 사용하여 조사하였다: 우선 $0.2{\mu}g/ml$의 매우 저농도에서 $200{\mu}g/ml$까지의 다양하게 희석한 엽록소 용액을 사용하여, 645 nm와 663 nm에서 측정한 흡광도 값을 측정한 후, 흡광도 값과 엽록소 농도의 비례관계를 조사하였다. 그 결과, 이 비례관계로 판단한 측정치의 신뢰 범위에서 흡광도로부터 계산한 용액에서의 엽록소 농도와 이미 알고 있는 엽록소 농도 비교적 일치하였다. 그러나, 비례관계의 한계치 안에 있어 신뢰도가 인정된 일부 고농도와 저농도에서의 일부 값이 엽록소 a/b 비례치가 용액의 희석에 변화지 않을 것이라는 판단 기준에 있어 신뢰도가 떨어짐을 알 수 있었다. 그러므로, 본 연구결과는 엽록소 농도결정을 위하여 측정한 흡광도 값의 신뢰 여부는 대상 용액을 희석하여 측정한 흡광도의 비례관계로 본 비어-램버트 법칙의 기준 보다는 엽록소 a/b 비례치가 변하지 않는다는 기준으로 측정치의 신뢰도를 판단하는 것이 더 좋다는 것을 확인할 수 있었다.

Fig. 1. Dependence of absorbance values on chlorophyll concentration using 80% acetone extracts in a relatively high chlorophyll concentration range from rice leaves. Absorbances were measured at 646.6 nm (A, B) or 646 nm (C, D) and at 663.6 nm (E, F) or 663 nm (G. H), using spectrophotometers: Cary4E (A, E), UV-1650PC (B, F), Versamax (C, G) and NanoDrop 1,000 (D, H). Experiments were repeated at least three times.

Fig. 2. Dependence of absorbance values on chlorophyll concentration using 80% acetone extracts in a relatively low chlorophyll concentration range from rice leaves. Absorbances were measured at 646.6 nm (A, B) or 646 nm (C, D) and at 663.6 nm (E, F) or 663 nm (G. H), using spectrophotometers: Cary4E (A, E), UV-1650PC (B, F), Versamax (C, G) and NanoDrop 1000 (D, H). Experiments were repeated at least three times except for C, D, G and H.

Fig. 3. Comparison of chlorophyll concentrations calculated based in the absorbance values measured with the known chlorophyll concentrations of solutions made by dilution of concentrated extracts. Chlorophyll concentrations calculated using solutions at a relatively low chlorophyll concentration range (A, B, C, D), and those calculated using solutions at a relatively high chlorophyll concentration range (E, F, G, H), using spectrophotometers: Cary4E (A, E), UV-1650PC (B, F), Versamax (C, G) and NanoDrop 1000 (D, H). Experiments were repeated at least three times except for C and D.

Fig. 4. Fidelity test based on the uniformity of chlorophyll a/b ratio in chlorophyll solutions diluted at various concentrations. Chlorophyll a/b ratios calculated using solutions at a relatively low chlorophyll concentration range (A, B, C, D), and those calculated using solutions at a relatively high chlorophyll concentration range (E, F, G, H), using spectrophotometers: Cary4E (A, E), UV-1650 PC (B, F), Versamax (C, G) and NanoDrop 1,000 (D, H). The dotted lines are given at 3.33 +/- 0.069, which is the average +/- standard deviation calculated using the 4 values from the 2^nd to 5^th chlorophyll a/b ratios from the left in E. Experiments were repeated at least three times except for C and D.