Key Word

least square method, calibration curve, blank solution, matrix, matrix effect, standard addition, internal standard

5 장 Calibration Method

* Calibration Curve : 분석물질의 감응도 곡선

Ex) page 92: 플로르 이온 전극감응도 곡선

How do you prepare a calibration curve ?

Martin Frant lab note

: Orion 사 제작 : 최초 F이온 전극에 대한 감응도

X 축 : mV

Y 축 : [F-]

5-1. Least Square Method for Finding the Best Straight Line

직선함수 수식 y = m x + b

m : 식 (5-3)

b : 식 (5-4)

y = 0.61538 x + 1.34615

유효숫자 ?

Construction of calibration curve:

1 prepare known samples of analyte covering a range of concentrations, and measure the response of the analytical procedure to these standards

Standard solutions solutions containing known concentrations of analyte

Blank solutions solutions containing all the reagents and solvent used in the analysis except the analyte

Conc A Conc B Conc C Conc D Conc E

-Prepare a blank solution and measure its response to the analytical procedure

- Subtract the average absorbance of the blank samples from each measured absorbance. This gives the corrected absorbance.

- Draw a graph of corrected absorbance versus quantity of protein.

Table 4-2: spectro data

- Find the best straight line through the line portion of the data

- When analysing an unknown solution in future, run a blank solution at the same time. Subtract the new blank absorbance from the absorbance of the unknown solution to obtain the correct absorbance. Use calibration curve to extrapolate the concentration of the unknown solution

Constructing a Calibration Curve

1. Prepare known samples of analyte, covering a convenient range of concentration, and measure the response of the analytical procedure to these standards.

2. Measure the response of the analytical procedure on the blank sample and subtract this value from the measured responses to obtain corrected responses.

3. Make a plot of corrected response versus quantity of analyte.

4. Obtain a best fit line (or curve) through the data. How do I do this?

5-2. 검량 ( 곡 ) 선< 작성절차 > 1. 표준물질 Response 측정 2. 바탕 보정 (Blank Correction) 3. 그래프 작성 , LSF 수식유도 및 오차분석

y = m x + b

Calibration Uncertainty:

x = [y(sy) - b(sb)]/m(sm) 그림 5-4 : 중앙에서 벗어날수록 불확도가 커짐 .

교재절차

식 5-15 가 더 정확함 : Sheet 사용

Yonsei Univ.

Uncertainties in Least Square’s fits

Each of our data points in our graph contains some uncertainty.

This uncertainty propagates into the slope and intercept.

When we use our calibration curve to determined the concentration of our unknown, the uncertainty in our unknown concentration is related to the uncertainty in the fit.

5-3 표준물질 첨가법 (Standard Addition)

1. 미지시료에 분석물질 첨가 : 분석물질 신호 증가도

입수

2. 미지시료 농도 결정

3. 주 용도 : 복잡한 매질 , 알지 못하는 매질의 시료

분석

예 ) 원자방출법에 의한 혈액중 Ca 정량

1. 물리적 영향 ( 시료주입속도 , 분무효율 )

2. 화학적 영향 ( 내열물질 형성 ) Yonsei Univ.

주 목적 : 매질효과 배제 방법 : 매질 매칭 (Matching) ?

매 질 : Everything besides analyte

초기 분석물질 농도

나중 분석물질 + 표준물질 농도=

초기용액 신호크기

나중용액 신호크기

수식 5-16

교재 중 예제풀이

그래프 방법 : 그림 5-5

Flask 1 contains no added standardFlask 2 contains 0.0200 M standardFlask 3 contains 0.0400 M standardFlask 4 contains 0.0600 M standardFlask 5 contains 0.0800 M standard

The standard is 0.200 M

그래프 방법 : 그림 5-6

5-4 내부표준물질 (Internal Standard)

내부표준물질 : 미지시료에 첨가하는 analyte 와는 다른 기준물질 신호비교 ---> 정량

용도 : 시료량이나 기기 감응도 변동시

예 1) 감응도 변동 : Chromatography 법 수 % 유량변동 ---> 수 % 감응도 변동 Analyte, standard 변동은 같음 : 그림 5-7

예 2) 시료주입량 변동 : GC 법 수 ul 주입시 주입량 변동 심함

방법 : 감응도비 결정

IS 물질의 조건 : 이상적인 IS: Isotope Dilution

Yonsei Univ.

Internal Standards An internal standard is a known amount of compound,

different from analyte that is added to the unknown. Signal from the analyte is compared with signal from

the internal standard to find out how much analyte is present.

standard ofion concentrat

signal standard of area

analyte ofion Concentrat

signal analyte of AreaF

F = response factor

Done with CH5