J. Sci.Fd Agric.

1974,25,237-245

The Mineral Composition of Apples

XI. An Extraction Technique Suitable for the Rapid Determination of
Calcium, but not Potassium and Magnesium, in the Fruit"
Michael A. Perring

East Malling Research Station, East Mulling, Maidstone, Ken f, England

(Manuscript received 26 March 1973 and accepted 6 November 1973)

Practically all the calcium could be extracted from apple tissue, provided that
particle size was reduced initially by a blending procedure, by heating with 6 N-
hydrochloric acid for 10 min. Tests with commercial samples indicated that
including the time required to remove the seeds, a determination of calcium could
be completed within 45 min of receiving a sample of 20 apples.
Potassium, phosphorus and hydrochloric acid were the main factors interfering
with the determination of 0.25 to 1.25 parts/million of calcium in solutions similar
to those of apple ash or extracts by emission in a flame spectrophotometer.
Average proportions of these and the other main constituents of the ash were
added to standard calcium solutions to overcome interferences in routine
analyses after dry ashing or extraction with hydrochloric acid.
Recoveries of potassium and magnesium were low when the extraction pro-
cedure was used.

1. Introduction
Low concentrations of calcium in apples have been associated with the susceptibility of
the fruit to various disorders in the orchard and in storage.1,2The storage of unsuitable
fruit could be avoided if calcium concentrations were known, but for this purpose a
rapid method for the measurement of calcium is necessary. The method described
previously3 was slow and consisted of four interdependent stages :(a) thorough blending
of the apple tissue, (b) ashing or digestion of a sub-sample, (c) separation of calcium
from interfering constituents, and (d) measurement of the calcium concentration. The
use of a flame spectrophotometer for stage (d) made stage (c) unnecessary. This is
discussed more fully below. The rate of analysis at stage (d) was increased further by
using an automated continuous flow system to dilute the solutions of the ash and feed
the spectrophotometer. The rate at stage (a) was increased by substituting the method
of blending apple tissue with water4 for freezing and grating.' Thus stage (b) became the
rate-determining step in the procedure.
It was thought that rapid digestions could be made with an automatic Kjeldahl
digester and that calcium could be measured simultaneously with nitrogen, potassium
Part X in this series is Perring, M. A. J . Sci. Fd. Agric. 1969,20,527,
9 237
238 M. A. Perring

and phosphorus. Some technical difficulties were encountered, but a method of ex-
traction with hydrochloric acid was tested and found to be effective. The procedure was
based on that of Premi and Cornfield6 who extracted Cu, Zn, Fe, Mn and Cr from plant
tissue and determined their concentrations with an atomic absorption spectrophoto-
meter.

2. Experimental
2.1. Samples
The extraction procedure was tested on bulked samples of whole Cox’s Orange Pippin
apples which had been frozen and grated without seeds or stalks at -20 “C5
More than a hundred samples of commercially grown Cox’s Orange Pippin apples
were analysed by the complete rapid procedure. Opposite pairs of quarters (including
seeds) were cut longitudinally from sub-samples of 20 apples, bulked and blended
with water. Seeds were removed from the remaining quarters of 38 of these samples and
they were frozen and grated at -20 “C.
Fine suspensions of apple in water were made from 30 to 40 g of frozen tissue by the
addition of 1 or 1.5 times the weight of distilled water followed by mixing in a laboratory
mixer/emulsifier (Silverson Machines Ltd) for 2 min. Opposite quarters of apples were
blended initially with distilled water in a commercial blender (Waring Products Corp.)
for 2 min and about 130 cm3 of the mixture was homogenised in the mi~er/emulsifier.~
2.2. Extraction and ashing
Extractions were made with varying amounts of acid as outlined below, but in the
finalised procedure 2.5 cm3 of a 1 : 1.5 apple to water mixture was syringed into a 25 cm3
conical flask and 1.25 cm3 concentrated hydrochloric acid (36 %, A.R.) was added. A
small funnel was placed in the neck of the flask which was put on a hot-plate that was
sufficiently hot to bring the contents of the flask to boiling point in 3 to 4 min and to
keep the liquid boiling gently for the remainder of a 10-min period. The liquid was
swirled occasionally during heating to wet tissue deposited on the sides of the flask. The
funnel and sides of the flask were rinsed with distilled water and the contents were
transferred to a conical, graduated centrifuge tube, made to 10 cm3 volume with
distilled water, stoppered and shaken vigorously. After centrifuging for 10 minutes at
2000 g portions of the supernatant liquid were transferred to sample cups.
Sub-samples of about 5 g of the frozen and grated fruit were dry ashed in platinum
crucibles5 Ash was dissolved by boiling with 5 cm3 1 N-hydrochloric acid. The solution
was transferred to a 20 cm3 standard flask and made to volume with distilled water.
2.3. Analysis
Solutions of extracted tissue or ash were sampled automatically, diluted approximately
1 : 10 or 1 :20, respectively, with distilled water and fed continuously to a flame spectro-
photometer (SP 900, Pye-Unicam Ltd) by means of Auto-Analyzer modules (Technicon
Instrument Co. Ltd) at the rate of 60 samples per hour. Appropriate standards were
included with each batch of samples. Care was taken to ensure that amounts of hydro-
chloric acid in the standards were similar to those in the samples and that the standards
Determination of calcium in apples 239

contained average proportions of the main constituents of the ash. This was achieved by
making compensating solutions containing potassium, phosphorus, magnesium,
sulphur and sodium in the approximate ratios 150: 15:5 :7 :2 and the appropriate
amounts of hydrochloric acid. Portions of these were diluted together with varying
amounts of strong calcium standards (to cover the range 2 to 10 in proportion to 150
units of potassium) to make the standard solutions used in the above procedures. For
example, the compensating solution for the analysis of solutions of ash of Cox’s Orange
Pippin apples was prepared as follows from “Specpur” chemicals (Johnson, Matthey
& Co. Ltd) : potassium carbonate, 2320 mg ; potassium dihydrogen orthophosphate,
659 mg; sodium chloride, 51 mg; magnesium sulphate (7H,O), 506 mg dissolved in
water and 88 cm3 of the batch of concentrated hydrochloric acid used (diluted) for
dissolving ash, were made to 500 cm3with distilled water. Amounts of calcium (calcium
carbonate dissolved in hydrochloric acid) ranging from 0.1 to 0.5 mg were put into 20
cm3 standard flasks, 2.5 cm3 of compensating solution was added to each flask and the
solutions were made to volume with distilled water.
Measurements of calcium (0.25 to 1.25 parts/million at the instrument) were made
with an acetylene (9-cm manometer reading) and compressed air (28 lb/in2) flame
produced by a Meker type of burner, at a wavelength of 422.7 nm and with a slit width of
0.08 mm. Results were recorded as peaks by a potentiometric recorder (Servoscribe).
Potassium determinations were made with a flame photometer (Evans Electro-
selenium Ltd) after an appropriate dilution with water. Hydrochloric acid was added to
the standard solutions to make them similar to those of apple extracts.
All glassware was of borosilicate glass to avoid contamination and the acidic stand-
ards and samples were not poured into the polystyrene sample cups until the analysis
of a batch was about to begin.

3. Results
3.1. Measurement of calcium
Several experiments were made on the measurement of calcium with the SP 900 flame
spectrophotometer. It was found that the baseline generally drifted slightly up or down,
but that the drift was regular. Compensation could be made by drawing the baseline
from those obtained at both ends of the series ofpeaks on the recorder chart and measur-
ing the heights of the peaks above the line.
3.2. Extraction of calcium
The results for a series of samples extracted by the above procedure (Section 2.2.) are
shown in Figure l(a). Reproducibility was good despite the amounts of apple extracted
(0.8 g) being small, and differences in calcium concentrations determined after extrac-
tion and dry ashing were negligible. This procedure was adopted after the three experi-
ments on extracting calcium from apple tissue described below had been made.
It was found that practically all of the calcium could be extracted by heating sub-
samples of frozen/grated apple with hydrochloric acid, diluted to bring its strength in
the mixtures to about 6 N, for 10 min. About 2 g of frozen/grated apple was heated with
acid, diluted to 100 cm3 after cooling, centrifuged and then fed directly to the flame
140 M. A. Perring
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Figure 1. Calcium concentrations found after extraction of apple tissues (without seeds) with
hydrochloric acid in relation to those found after dry ashing.
Procedures: (a) 2 cm3 1 apple: 1.5 water suspension extracted, 10 cm3 centrifuged then diluted.
Analyses duplicated: first extraction X; second extraction 0.(b) 2-g of apple extracted, diluted to 100
c1n3 then centrifuged. One extraction. duplicated analyses X. (c) 2.5 g of apple extracted, 20 cm3
centrifuged then diluted. Means of duplicated analyses: first extraction X; second extraction 0 ; third
extraction :. (d) 5 cm3 1 apple: 1 water extracted, 20 cm3 centrifuged then diluted. Means ofduplicated
analyses: first extraction X ; second extraction 0 .

spectrophotometer. Recoveries of calcium were slightly high in comparison with

results obtained by ashing but results of duplicated determinations of calcium in the
centrifuged solution were in good agreement [Figure 1 (b)].
As the risk of contamination of the samples would be high in routine analysis, a
technique of centrifuging followed by dilution in a closed system was investigated. The
amount of solids separated in the centrifuge was not negligible as in the previous
procedure, but amounted to about a tenth of the volume. Results were therefore
corrected by a factor which depended on the amount of apple extracted and was assessed
from the calibrated centrifuge tubes. Results for a series of 2.5 g sub-samples of frozen/
grated apple which were made t o 20 cm3 volume after extraction and cooling showed
that the amount of calcium recovered was not very reproducible [Figure I(c)]. This
appeared to be due to the fairly coarse particles in the gratcd tissue and so samples were
blended with an equal weight of water for two minutes in a mixer/emulsifier4 before the
extraction procedure. Reproducible results were obtained in this way and differences in
calcium concentrations determined after extraction and dry ashing were negligible
[Figure I(d)]. However, when apples that had been stored at 0 'C were blended with
water in the ratio 1 : 1 for analysis by the extraction procedure for calcium and the auto-
mated Kjeldahl system for nitrogen, it was found that some samples gelled in the cups
and could not be pumped to the Kjeldahl digestor. Hence the ratio of water to apple
had to be increased to 1.5 : I to overcome this problem.
Recoveries of calcium were apparently about 10 7; high, in relation t o the theoretical
line shown in Figure 2, when results of extractions of bulked samples of apples were
Determination of calcium in apples 241

n 0

compared with results obtained by dry ashing. However, the seeds, which are high in
c a l c i ~ mwere
, ~ left in the apple tissue that was extracted and the variability in recoveries,
attributable to the seeds, is more noteworthy.

3.3. Extraction of potassium and magnesium

Potassium was determined in the extracts of the commercial samples (and also in
extracts of frozen/grated samples) and with one exception recoveries were found to be
low and very variable (Figure 2). Recoveries of magnesium were as low in the few samples
for which measurements were made by the atomic absorption technique as those of
potassium.

4. Discussion
4.1. Measurement of calcium
Although the measurement of calcium with the SP 900 flame spectrophotometer has
been described in some detail because many analyses of calcium have been made with
this instrument since 1965, the discussion will be confined mainly to points generally
applicable to the measurement of calcium in apples by flame techniques. Results were
found to be slightly lower (approx. 5 %) than those obtained after precipitation of the
calcium as ~ x a l a t eThis
. ~ was probably due to slight increases in calcium concentrations
in oxalate precipitates caused by handling and successive additions of very small
quantities of calcium as impurities in reagents. Attempts to measure the small “blank”
values involved were unsuccessful. Drifting of calibration should not occur in modern
double-beam absorption instruments and breakdowns due to attack on the stainless
242 M. A. Perring

steel of the burner by the relatively strong hydrochloric acid in the extracts could be
overcome by using titanium burners and nebuliser units coated with fluorocarbons.
The effect of potassium of increasing readings diminishes with increasing concentra-
tions (Table I), so errors due to potassium in the samples deviating from the mean of
150 mg per 100 g used in the compensating solution would be very small in the range
found in Cox’s Orange Pippin apples. Jones and Thomas, who added increasing
amounts of sodium phosphate to standard calcium solutions, found no increase in
interferences with P :Ca ratios from 5 : 1 to 750: 1 when measurements were made with
an SP 900 spectrophotometer.’ However, phosphate in the range likely to be found in
apples (P:Ca, 1 : I to 20: 1) depressed chart readings by 6 to 9 when the effect of
potassium in the phosphate added to standard calcium solutions was taken into

TABLE1. Chart readings (means of 3) obtained by the flame emission technique

when potassium (as KCI) was added to standard calcium solutions in ratios similar
to those found in Cox’s Orange Pippin apples

Potassium

Calcium 0 12.5 parts/million (100) 25 parts/million (200)

0.25 parts/million (2) 16.7 20.7 22.7

0.75 parts/million (6) 50.5 62.2 64.3

Figures in brackets are equivalent values in mg per 100 g fresh weight.

TABLE 2. Chart readings (means of 2) obtained by the flame emission technique when hydrochloric
acid, equivalent in amounts to that in the compensating solution and that used in extracting calcium
from apple tissue; and compensating solution, with the additional amount of hydrochloric acid used in
extractions, were added in appropriate proportions to standard calcium solutions

Additive

Hydrochloric acid, Compensating solution + Hydrochloric acid,

Calcium amount used in extra hydrochloric acid amount used in
(parts/million) compensating yolution used in extraction extraction

0.25 44.5 42.0 38.0

0.50 86.8 81.3 79.0

consideration (Table 3). The only other interfering constituent was the hydrochloric
acid (Table 2), but it was considered safer to include average amounts of all the major
constituents because it had been found in previous experiments on interferences in
determinations of sodium in a cooler flame that the combined effect of all the con-
stituents was not the same as that expected from those found indi~ idually;~ and
Bradfield and Spincer noted interferences due to mixtures of magnesium and sulphate
in the measurement of zinc by the atomic absorption rnethod.’O
The compensating solution described is for Cox’s Orange Pippin apples. Compensat-
ing solutions containing different proportions of constituents are necessary for the
Determination of calcium in apples 243

TABLE3. Chart readings (means of 2) obtained by the flame emission

technique when phosphorus (KH2P04),and potassium (KCI), in amounts
equivalent to those in the phosphate (readings shown in brackets), were
added to standard calcium solutions.
_ _ _ _ _ ~
~~~ ~ ~

Phosphorus, amounts in proportion to those of calcium

Calcium I

(parts/million) 0 lox 20x

0.25 16.5 18.0 19.6

- (19.8) (20.8)
0.75 47.0 50.2 52.6
- (53.1) (58.0)

determination of calcium in other varieties; e.g. for Bramley’s Seedling potassium,

phosphorus and magnesium concentrations in the ratios 100: 10:4for the range 2 to 10
of calcium are more appropriate.
Extra calcium was found in one batch of extracts stored overnight in covered poly-
styrene sample cups. This was apparently absorbed from the cups by the acid and
considerable amounts of calcium were found when new cups were dry ashed. In further
tests it was found that extracts could be left overnight, if necessary, in the borosilicate
glass centrifuge tubes.
4.2. Extraction of calcium
The results of analyses of sub-samples of frozen/grated apples from which the seeds
had been removed (Figure 1) showed that practically all of the calcium could be
extracted from apple tissue by boiling for a short time with hydrochloric acid and that
various methods of dilution of the extracts could be employed. Differences in results of
replicated extractions when the apple had been suspended finely in water were of the
same order (Table 4) as those incurred in duplicating analyses of the same extract with
the flame spectrophotometer, and in the sub-sampling, ashing and analysis of 5 g of
frozen/grated apple (coefficient of variation = 2.65).
When the extraction procedure was used in conjunction with sample preparation by
blending apples with water, the calcium concentration in a sample of 20 apples could
be obtained by one worker within 35 min if the seeds were not removed. However, it
was found that variations in apparent recoveries of calcium from individual samples

TABLE 4. Coefficients of variation found when 10 sub-samples of about 3 cm3 of

1 :1.5 apple (including seeds) : water suspensions were extracted with hydrochloric
acid and calcium was determined with the flame spectrophotometer

Range of calcium concentrations

(mg per 100 g of fresh weight) Mean calcium Coefficient of
Sample in 10 sub-samples concentration variation

0 21 6.20-6.50 , 6.25 1.47

s 20 3.85-4.10 3.95 2.04
WK 12 3.90-4.30 4.09 2.98

Each calcium result was the mean of two determinations with the instrument.
244 M. A. Perring

were large when the combined procedure was tested on commercial samples (Figure 2).
Corrections by a single factor for the increase in concentration due to calcium in the
seeds, and hence the accurate prediction of storage disorders, were impossible. Further
experiments confirmed the necessity of removal of seeds’ and so, with the time for
this included, the analysis of a single sample would take about 45 min which is much
quicker than any procedure involving wet or dry ashing and comparable with the time
required for automated Kjeldahl digestions and analyses. Calculation of results would
be unnecessary in the rapid assessment of storage potential if standards used corres-
ponded with the threshold values for various disorders and sub-samples of suspensions
were measured accurately by volume.
Apart from the prediction of storage disorders, determinations by the rapid procedure
would be of great value in studying calcium in individual apples because the range of
concentrations is large, and in screening samples from experimental trials so that those
of interest could be selected for analysis by other methods.
Two of the samples, with calcium concentrations of about 10 and 6.5 mg/100 g
[Figure l(a)], were picked at the ends of July and August. The results indicate that
calcium can be recovered from immature apples by extraction as effectivelyas from those
picked at normal times.

4.3. Recoveries of potassium and magnesium

A knowledge of potassium concentrations is valuable in estimating the severity of
lenticel blotch pit and bitter pit and samples of apples with low levels of magnesium are
more susceptible to low temperature breakdown.’, l 1 It would have been convenient
to divide the extracts at the analytical stage and so determine potassium and magnesium
simultaneously with calcium, but this could not be done because of the low and variable
recoveries of potassium (Figure 2) and magnesium. Some potassium determinations
were repeated with the hotter acetylene flame in the spectrophotometer but results were
no higher than those obtained with the coal-gas flame in the simpler instrument. It is
probable that about a tenth of the potassium and magnesium was retained in the black
residue. It was found recently that recoveries ofpotassium and magnesium from samples
incompletely dry ashed and containing considerable amounts of carbon were low,
whereas those of calcium were Basson and BOhmerl3recorded low recoveries
of potassium and magnesium when leaf tissue was extracted with 6 M-hydrochloric acid
and overcame the problem by reducing the concentration to 3 M. No experiments have
been made on extracting fruit tissue with lower concentrations of acid but most of the
potassium (95 to 98 2)can be extracted with water3 and analyses could be made in this
way by dilution after the blending processes. An estimate of magnesium levels could
be made from those of potassium because it has been shown that there is a roughly
h e a r relationship between them in bulked samples of fruit from orchard trials’4, l 5 and
commercial sources.16
Acknowledgements
Mrs I. Cook assisted with the development of the extraction procedure and used it to
determine calcium and potassium in the commercial samples and Mr K. Pearson
assisted with the analyses of these samples by the dry ashing technique.
Determination of calcium in apples 245

References
1. Perring, M. A. J. Sci. Fd Agric. 1968, 19, 186.
2. Perring, M. A. J. Sci. Fd Agric. 1968, 19, 640.
3. Perring, M. A. J . Sci. Fd Agric. 1964, 15,752.
4. Perring, M. A. J. Sci. FdAgric. 1969,20, 527.
5. Perring, M. A. J. Sci. Fd Agric. 1964, 15, 739.
6. Premi, P. R. ; Cornfield, A. H. Spectrovision 1968, 19, 15.
7. Perring, M. A. J . Sci. Fd Agric. 1974,25, 247.
8 . Jones, J. G .; Thomas, J. D. R. Analyst, Lond. 1966, 91, 559.
9. Perring, M. A. J. Sci.Fd Agric. 1967, 18, 265.
10. Bradfield, E. G.; Spincer, D. J. Sci. Fd Agric. 1965, 16, 33.
11. Perring, M. A. Rep. E. Mulling Rex Stn for 1967, 1968, p. 191.
12. Perring, M. A. Rep. E. Malting Res. Stn for 1972 (1973) p. 112.
13. Basson, W. D. ; Bohmer, R. G. Analyst, Lond. 1972,97,482.
14. Wilkinson, B. G. J. hort. Sci. 1958,33, 49.
15. Perring, M. A. ; Preston, A. P. J. hort. Sci. 1974,49 (In the press).
16. Perring, M. A.; Sharples, R. 0. Unpublished results. 1965 to 1970.